US20170007679A1 - Crispr/cas-related methods and compositions for treating hiv infection and aids - Google Patents

Abstract

CRISPR/CAS-related compositions and methods for treatment of a subject at risk for or having a HIV infection or AIDS are disclosed.

Description

REFERENCE TO RELATED APPLICATIONS
• This application is a continuation of PCT International Patent Application No. PCT/US2015/022497, filed on Mar. 25, 2015, which claims the benefit of U.S. Provisional Application No. 61/970,237, filed Mar. 25, 2014, the contents of each of which are hereby incorporated by reference in their entirety herein, and to each of which priority is claimed.
SEQUENCE LISTING
• The specification further incorporates by reference the Sequence Listing submitted herewith via EFS on Sep. 23, 2016. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified as 084177.0124SEQ.txt, is 2,093,238 bytes and was created on Sep. 23, 2016. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter.
FIELD OF THE INVENTION
• The invention relates to CRISPR/CAS-related methods and components for editing of a target nucleic acid sequence, and applications thereof in connection with Human Immunodeficiency Virus (HIV) infection and Acquired Immunodeficiency Syndrome (AIDS).
BACKGROUND
• Human Immunodeficiency Virus (HIV) is a virus that causes severe immunodeficiency. In the United States, more than 1 million people are infected with the virus. Worldwide, approximately 30-40 million people are infected.
• HIV preferentially infects CD4 T cells. It causes declining CD4 T cell counts, severe opportunistic infections and certain cancers, including Kaposi's sarcoma and Burkitt's lymphoma. Untreated HIV infection is a chronic, progressive disease that leads to acquired immunodeficiency syndrome (AIDS) and death in nearly all subjects.
• HIV was untreatable and invariably led to death in all subjects until the late 1980's. Since then, antiretroviral therapy (ART) has dramatically slowed the course of HIV infection. Highly active antiretroviral therapy (HAART) is the use of three or more agents in combination to slow HIV. Treatment with HAART has significantly altered the life expectancy of those infected with HIV. A subject in the developed world who maintains their HAART regimen can expect to live into his or her 60's and possibly 70's. However, HAART regimens are associated with significant, long-term side effects. The dosing regimens are complex and associated with strict dietary requirements. Compliance rates with dosing can be lower than 50% in some populations in the United States. In addition, there are significant toxicities associated with HAART treatment, including diabetes, nausea, malaise and sleep disturbances. A subject who does not adhere to dosing requirements of HAART therapy may have a return of viral load in their blood and is at risk for progression of the disease and its associated complications.
• HIV is a single-stranded RNA virus that preferentially infects CD4 T-cells. The virus must bind to receptors and coreceptors on the surface of CD4 cells to enter and infect these cells. This binding and infection step is vital to the pathogenesis of HIV. The virus attaches to the CD4 receptor on the cell surface via its own surface glycoproteins, gp120 and gp41. Gp120 binds to a CD4 receptor and must also bind to another coreceptor in order for the virus to enter the host cell. In macrophage-(M-tropic) viruses, the coreceptor is CCR5, also referred to as the CCR5 receptor. CCR5 receptors are expressed by CD4 cells, T cells, gut-associated lymphoid tissue (GALT), macrophages, dendritic cells and microglia. HIV establishes initial infection and replicates in the host most commonly via CCR5 co-receptors.
• As most HIV infections and early stage HIV is due to entry and propagation of M-tropic virus, CCR5-Δ32 mutation results in a non-functional CCR5 receptor that does not allow M-tropic HIV-1 virus entry. Individuals carrying two copies of the CCR5-Δ32 allele are resistant to HIV infection and CCR5-Δ32 heterozygous carriers have slow progression of the disease.
• CCR5 antagonists (e.g. maraviroc) exist and are used in the treatment of HIV. However, current CCR5 antagonists decrease HIV progression but cannot cure the disease. In addition, there are considerable risks of side effects of these CCR5 antagonists, including severe liver toxicity.
• In spite of considerable advances in the treatment of HIV, there remain considerable needs for agents that could prevent, treat, and eliminate HIV infection or AIDS. Therapies that are free from significant toxicities and involve a single or multi-dose regimen (versus current daily dose regimen for the lifetime of a patient) would be superior to current HIV treatment. A reduction or complete elimination of CCR5 expression in myeloid and lymphoid cells would prevent HIV infection and progression, and even cure this disease.
SUMMARY OF THE INVENTION
• Methods and compositions discussed herein, allow for the prevention and treatment of HIV infection and AIDS, by introducing one or more mutations in the gene for C-C chemokine receptor type 5 (CCR5). The CCR5 gene is also known as CKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, IDDM22, and CC-CKR-5.
• Methods and compositions discussed herein, provide for prevention or reduction of HIV infection and/or prevention or reduction of the ability for HIV to enter host cells, e.g., in subjects who are already infected. Exemplary host cells for HIV include, but are not limited to, CD4 cells, T cells, gut associated lymphatic tissue (GALT), macrophages, dendritic cells, myeloid precursor cell, and microglia. Viral entry into the host cells requires interaction of the viral glycoproteins gp41 and gp120 with both the CD4 receptor and a co-receptor, e.g., CCR5. If a co-receptor, e.g., CCR5, is not present on the surface of the host cells, the virus cannot bind and enter the host cells. The progress of the disease is thus impeded. By knocking out or knocking down CCR5 in the host cells, e.g., by introducing a protective mutation (such as a CCR5 delta 32 mutation), entry of the HIV virus into the host cells is prevented.
• Methods and compositions discussed herein, provide for treating or delaying the onset or progression of HIV infection or AIDS by gene editing, e.g., using CRISPR-Cas9 mediated methods to alter a CCR5 gene. Altering the CCR5 gene herein refers to reducing or eliminating (1) CCR5 gene expression, (2) CCR5 protein function, or (3) the level of CCR5 protein.
• In one aspect, the methods and compositions discussed herein, inhibit or block a critical aspect of the HIV life cycle, i.e., CCR5-mediated entry into T cells, by alteration (e.g., inactivation) of the CCR5 gene. Exemplary mechanisms that can be associated with the alteration of the CCR5 gene include, but are not limited to, non-homologous end joining (NHEJ) (e.g., classical or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (e.g., endogenous donor template mediated), SDSA (synthesis dependent strand annealing), single strand annealing or single strand invasion. Alteration of the CCR5 gene, e.g., mediated by NHEJ, can result in a mutation, which typically comprises a deletion or insertion (indel). The introduced mutation can take place in any region of the CCR5 gene, e.g., a promoter region or other non-coding region, or a coding region, so long as the mutation results in reduced or loss of the ability to mediate HIV entry into the cell.
• In another aspect, the methods and compositions discussed herein may be used to alter the CCR5 gene to treat or prevent HIV infection or AIDS by targeting the coding sequence of the CCR5 gene.
• In an embodiment, the gene, e.g., the coding sequence of the CCR5 gene, is targeted to knock out the gene, e.g., to eliminate expression of the gene, e.g., to knock out both alleles of the CCR5 gene, e.g., by introduction of an alteration comprising a mutation (e.g., an insertion or deletion) in the CCR5 gene. This type of alteration is sometimes referred to as “knocking out” the CCR5 gene. While not wishing to be bound by theory, in an embodiment, a targeted knockout approach is mediated by NHEJ using a CRISPR/Cas system comprising a Cas9 molecule, e.g., an enzymatically active Cas9 (eaCas9) molecule, as described herein.
• In another aspect, the methods and compositions discussed herein may be used to alter the CCR5 gene to treat or prevent HIV infection or AIDS by targeting a non-coding sequence of the CCR5 gene, e.g., a promoter, an enhancer, an intron, a 3′UTR, and/or a polyadenylation signal.
• In one embodiment, the gene, e.g., the non-coding sequence of the CCR5 gene, is targeted to knock out the gene, e.g., to eliminate expression of the gene, e.g., to knock out both alleles of the CCR5 gene, e.g., by introduction of an alteration comprising a mutation (e.g., an insertion or deletion) in the CCR5 gene. In an embodiment, the method provides an alteration that comprises an insertion or deletion. This type of alteration is also sometimes referred to as “knocking out” the CCR5 gene. While not wishing to be bound by theory, in an embodiment, a targeted knockout approach is mediated by NHEJ using a CRISPR/Cas system comprising a Cas9 molecule, e.g., an enzymatically active Cas9 (eaCas9) molecule, as described herein.
• In an embodiment, methods and compositions discussed herein, provide for altering (e.g., knocking out) the CCR5 gene. In an embodiment, knocking out the CCR5 gene herein refers to (1) insertion or deletion (e.g., NHEJ-mediated insertion or deletion) of one or more nucleotides of the CCR5 gene (e.g., in close proximity to or within an early coding region or in a non-coding region), or (2) deletion (e.g., NHEJ-mediated deletion) of a genomic sequence of the CCR5 gene (e.g., in a coding region or in a non-coding region). Both approaches give rise to alteration of the CCR5 gene as described herein. In an embodiment, a CCR5 target knockout position is altered by genome editing using the CRISPR/Cas9 system. The CCR5 target knockout position may be targeted by cleaving with either one or more nucleases, or one or more nickases, or a combination thereof.
• “CCR5 target knockout position”, as used herein, refers to a position in the CCR5 gene, which if altered, e.g., disrupted by insertion or deletion of one or more nucleotides, e.g., by NHEJ-mediated alteration, results in alteration of the CCR5 gene. In an embodiment, the position is in the CCR5 coding region, e.g., an early coding region. In another embodiment, the position is in a non-coding sequence of the CCR5 gene, e.g., a promoter, an enhancer, an intron, a 3′UTR, and/or a polyadenylation signal.
• In another embodiment, the CCR5 gene is targeted to knock down the gene, e.g., to reduce or eliminate expression of the gene, e.g., to knock down one or both alleles of the CCR5 gene.
• In one embodiment, the coding region of the CCR5 gene, is targeted to alter the expression of the gene. In another embodiment, a non-coding region (e.g., an enhancer region, a promoter region, an intron, a 5′ UTR, a 3′UTR, or a polyadenylation signal) of the CCR5 gene is targeted to alter the expression of the gene. In an embodiment, the promoter region of the CCR5 gene is targeted to knock down the expression of the CCR5 gene. This type of alteration is also sometimes referred to as “knocking down” the CCR5 gene. While not wishing to be bound by theory, in an embodiment, a targeted knockdown approach is mediated by a CRISPR/Cas system comprising a Cas9 molecule, e.g., an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain or chromatin modifying protein), as described herein. In an embodiment, the CCR5 gene is targeted to alter (e.g., to block, reduce, or decrease) the transcription of the CCR5 gene. In another embodiment, the CCR5 gene is targeted to alter the chromatin structure (e.g., one or more histone and/or DNA modifications) of the CCR5 gene. In an embodiment, a CCR5 target knockdown position is targeted by genome editing using the CRISPR/Cas9 system. In an embodiment, one or more gRNA molecules comprising a targeting domain are configured to target an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain), sufficiently close to a CCR5 target knockdown position to reduce, decrease or repress expression of the CCR5 gene.
• “CCR5 target knockdown position”, as used herein, refers to a position in the CCR5 gene, which if targeted, e.g., by an eiCas9 molecule or an eiCas9 fusion described herein, results in reduction or elimination of expression of functional CCR5 gene product. In an embodiment, the transcription of the CCR5 gene is reduced or eliminated. In another embodiment, the chromatin structure of the CCR5 gene is altered. In an embodiment, the position is in the CCR5 promoter sequence. In an embodiment, a position in the promoter sequence of the CCR5 gene is targeted by an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein, as described herein.
• “CCR5 target position”, as used herein, refers to any position that results in inactivation of the CCR5 gene. In an embodiment, a CCR5 target position refers to any of a CCR5 target knockout position or a CCR5 target knockdown position, as described herein.
• In one aspect, disclosed herein is a gRNA molecule, e.g., an isolated or non-naturally occurring gRNA molecule, comprising a targeting domain which is complementary with a target domain from the CCR5 gene.
• In an embodiment, the targeting domain of the gRNA molecule is configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a CCR5 target position in the CCR5 gene to allow alteration, e.g., alteration associated with NHEJ, of a CCR5 target position in the CCR5 gene. In an embodiment, the alteration comprises an insertion or deletion. In an embodiment, the targeting domain is configured such that a cleavage event, e.g., a double strand or single strand break, is positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 450, or 500 nucleotides of a CCR5 target position. The break, e.g., a double strand or single strand break, can be positioned upstream or downstream of a CCR5 target position in the CCR5 gene.
• In an embodiment, a second gRNA molecule comprising a second targeting domain is configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to the CCR5 target position in the CCR5 gene, to allow alteration, e.g., alteration associated with NHEJ, of the CCR5 target position in the CCR5 gene, either alone or in combination with the break positioned by said first gRNA molecule. In an embodiment, the targeting domains of the first and second gRNA molecules are configured such that a cleavage event, e.g., a double strand or single strand break, is positioned, independently for each of the gRNA molecules, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 450, or 500 nucleotides of the target position. In an embodiment, the breaks, e.g., double strand or single strand breaks, are positioned on both sides of a nucleotide of a CCR5 target position in the CCR5 gene. In an embodiment, the breaks, e.g., double strand or single strand breaks, are positioned on one side, e.g., upstream or downstream, of a nucleotide of a CCR5 target position in the CCR5 gene.
• In an embodiment, a single strand break is accompanied by an additional single strand break, positioned by a second gRNA molecule, as discussed below. For example, the targeting domains are configured such that a cleavage event, e.g., the two single strand breaks, are positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 450, or 500 nucleotides of a CCR5 target position. In an embodiment, the first and second gRNA molecules are configured such, that when guiding a Cas9 molecule, e.g., a Cas9 nickase, a single strand break will be accompanied by an additional single strand break, positioned by a second gRNA, sufficiently close to one another to result in alteration of a CCR5 target position in the CCR5 gene. In an embodiment, the first and second gRNA molecules are configured such that a single strand break positioned by said second gRNA is within 10, 20, 30, 40, or 50 nucleotides of the break positioned by said first gRNA molecule, e.g., when the Cas9 molecule is a nickase. In an embodiment, the two gRNA molecules are configured to position cuts at the same position, or within a few nucleotides of one another, on different strands, e.g., essentially mimicking a double strand break.
• In an embodiment, a double strand break can be accompanied by an additional double strand break, positioned by a second gRNA molecule, as is discussed below. For example, the targeting domain of a first gRNA molecule is configured such that a double strand break is positioned upstream of a CCR5 target position in the CCR5 gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 450, or 500 nucleotides of the target position; and the targeting domain of a second gRNA molecule is configured such that a double strand break is positioned downstream of a CCR5 target position in the CCR5 gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 450, or 500 nucleotides of the target position.
• In an embodiment, a double strand break can be accompanied by two additional single strand breaks, positioned by a second gRNA molecule and a third gRNA molecule. For example, the targeting domain of a first gRNA molecule is configured such that a double strand break is positioned upstream of a CCR5 target position in the CCR5 gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 450, or 500 nucleotides of the target position; and the targeting domains of a second and third gRNA molecule are configured such that two single strand breaks are positioned downstream of a CCR5 target position in the CCR5 gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 450, or 500 nucleotides of the target position. In an embodiment, the targeting domain of the first, second and third gRNA molecules are configured such that a cleavage event, e.g., a double strand or single strand break, is positioned, independently for each of the gRNA molecules.
• In an embodiment, a first and second single strand breaks can be accompanied by two additional single strand breaks positioned by a third gRNA molecule and a fourth gRNA molecule. For example, the targeting domain of a first and second gRNA molecule are configured such that two single strand breaks are positioned upstream of a CCR5 target position in the CCR5 gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 450, or 500 nucleotides of the target position; and the targeting domains of a third and fourth gRNA molecule are configured such that two single strand breaks are positioned downstream of a CCR5 target position in the CCR5 gene, e.g., within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 450, or 500 nucleotides of the target position.
• It is contemplated herein that, in an embodiment, when multiple gRNAs are used to generate (1) two single stranded breaks in close proximity, (2) two double stranded breaks, e.g., flanking a CCR5 target position (e.g., to remove a piece of DNA, e.g., a insertion or deletion mutation) or to create more than one indel in an early coding region, (3) one double stranded break and two paired nicks flanking a CCR5 target position (e.g., to remove a piece of DNA, e.g., a insertion or deletion mutation) or (4) four single stranded breaks, two on each side of a CCR5 target position, that they are targeting the same CCR5 target position. It is further contemplated herein that in an embodiment multiple gRNAs may be used to target more than one target position in the same gene.
• In an embodiment, the targeting domain of the first gRNA molecule and the targeting domain of the second gRNA molecules are complementary to opposite strands of the target nucleic acid molecule. In an embodiment, the gRNA molecule and the second gRNA molecule are configured such that the PAMs are oriented outward.
• In an embodiment, the targeting domain of a gRNA molecule is configured to avoid unwanted target chromosome elements, such as repeat elements, e.g., Alu repeats, in the target domain. The gRNA molecule may be a first, second, third and/or fourth gRNA molecule, as described herein.
• In an embodiment, the targeting domain of a gRNA molecule is configured to position a cleavage event sufficiently far from a preselected nucleotide, e.g., the nucleotide of a coding region, such that the nucleotide is not altered. In an embodiment, the targeting domain of a gRNA molecule is configured to position an intronic cleavage event sufficiently far from an intron/exon border, or naturally occurring splice signal, to avoid alteration of the exonic sequence or unwanted splicing events. The gRNA molecule may be a first, second, third and/or fourth gRNA molecule, as described herein.
• In an embodiment, a CCR5 target position is targeted and the targeting domain of a gRNA molecule comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any one of Tables 1A-1F, 2A-2C, 3A-3E, or 4A-4C. In an embodiment, the targeting domain is independently selected from those in Tables 1A-1F, 2A-2C, 3A-3E, or 4A-4C. In an embodiment, the targeting domain is independently selected from:

• (SEQ ID NO: 387)

  	CCUGCCUCCGCUCUACUCAC;

  (SEQ ID NO: 388)

  	GCUGCCGCCCAGUGGGACUU;

  (SEQ ID NO: 389)

  	ACAAUGUGUCAACUCUUGAC;

  (SEQ ID NO: 390)

  	GGUGACAAGUGUGAUCACUU;

  (SEQ ID NO: 391)

  	CCAGGUACCUAUCGAUUGUC;

  (SEQ ID NO: 392)

  	CUUCACAUUGAUUUUUUGGC;

  (SEQ ID NO: 393)

  	GCAGCAUAGUGAGCCCAGAA;

  (SEQ ID NO: 394)

  	GGUACCUAUCGAUUGUCAGG;

  (SEQ ID NO: 395)

  	GUGAGUAGAGCGGAGGCAGG;

  (SEQ ID NO: 396)

  	GCCUCCGCUCUACUCAC;

  (SEQ ID NO: 397)

  	GCCGCCCAGUGGGACUU;

  (SEQ ID NO: 398)

  	AUGUGUCAACUCUUGAC;

  (SEQ ID NO: 399)

  	GACAAUCGAUAGGUACC;

  (SEQ ID NO: 400)

  	CACAUUGAUUUUUUGGC;

  (SEQ ID NO: 401)

  	GCAUAGUGAGCCCAGAA;
  	or

  (SEQ ID NO: 402)

  GGUACCUAUCGAUUGUC.

• In an embodiment, the targeting domain is independently selected from those in Table 2A. In an embodiment, the targeting domain is independently selected from those in Table 3A. In an embodiment, the targeting domain is independently selected from those in Table 4A.
• In an embodiment, more than one gRNA is used to position breaks, e.g., two single stranded breaks or two double stranded breaks, or a combination of single strand and double strand breaks, e.g., to create one or more indels, in the target nucleic acid sequence. In an embodiment, the targeting domain of each guide RNA is independently selected from any one of Tables 1A-1F, 2A-2C, 3A-3E, or 4A-4C.
• In an embodiment, the targeting domain of the gRNA molecule is configured to target an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain), sufficiently close to a CCR5 transcription start site (TSS) to reduce (e.g., block) transcription, e.g., transcription initiation or elongation, binding of one or more transcription enhancers or activators, and/or RNA polymerase. In an embodiment, the targeting domain is configured to target between 1000 bp upstream and 1000 bp downstream (e.g., between 500 bp upstream and 1000 bp downstream, between 1000 bp upstream and 500 bp downstream, between 500 bp upstream and 500 bp downstream, within 500 bp or 200 bp upstream, or within 500 bp or 200 bp downstream) of the TSS of the CCR5 gene. One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.
• In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any one of Tables 5A-5C, 6A-6E, or 7A-7C. In an embodiment, the targeting domain is independently selected from those in Tables 5A-5C, 6A-6E, or 7A-7C.
• In an embodiment, the targeting domain is independently selected from those in Table 5A. In an embodiment, the targeting domain is independently selected from those in Table 6A. In an embodiment, the targeting domain is independently selected from those in Table 7A.
• In an embodiment, when the CCR5 promoter region is targeted, e.g., for knockdown, the targeting domain can comprise a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any one of Tables 5A-5C, 6A-6E, or 7A-7C. In an embodiment, the targeting domain is independently selected from those in Tables 5A-5C, 6A-6E, or 7A-7C.
• In an embodiment, when the CCR5 target knockdown position is the CCR5 promoter region and more than one gRNA is used to position an eiCas9 molecule or an eiCas9-fusion protein (e.g., an eiCas9-transcription repressor domain fusion protein), in the target nucleic acid sequence, the targeting domain for each guide RNA is independently selected from one of Tables 5A-5C, 6A-6E, or 7A-7C.
• In an embodiment, the targeting domain comprises a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from Table 18. In an embodiment, the targeting domain is independently selected from those in Table 18.
• In an embodiment, the targeting domain which is complementary with a target domain from the CCR5 target position in the CCR5 gene is 16 nucleotides or more in length. In an embodiment, the targeting domain is 16 nucleotides in length. In an embodiment, the targeting domain is 17 nucleotides in length. In other embodiments, the targeting domain is 18 nucleotides in length. In still other embodiments, the targeting domain is 19 nucleotides in length. In still other embodiments, the targeting domain is 20 nucleotides in length. In an embodiment, the targeting domain is 21 nucleotides in length. In an embodiment, the targeting domain is 22 nucleotides in length. In an embodiment, the targeting domain is 23 nucleotides in length. In an embodiment, the targeting domain is 24 nucleotides in length. In an embodiment, the targeting domain is 25 nucleotides in length. In an embodiment, the targeting domain is 26 nucleotides in length.
• In an embodiment, the targeting domain comprises 16 nucleotides.
• In an embodiment, the targeting domain comprises 17 nucleotides.
• In an embodiment, the targeting domain comprises 18 nucleotides.
• In an embodiment, the targeting domain comprises 19 nucleotides.
• In an embodiment, the targeting domain comprises 20 nucleotides.
• In an embodiment, the targeting domain comprises 21 nucleotides.
• In an embodiment, the targeting domain comprises 22 nucleotides.
• In an embodiment, the targeting domain comprises 23 nucleotides.
• In an embodiment, the targeting domain comprises 24 nucleotides.
• In an embodiment, the targeting domain comprises 25 nucleotides.
• In an embodiment, the targeting domain comprises 26 nucleotides.
• A gRNA as described herein may comprise from 5′ to 3′: a targeting domain (comprising a “core domain”, and optionally a “secondary domain”); a first complementarity domain; a linking domain; a second complementarity domain; a proximal domain; and a tail domain. In some embodiments, the proximal domain and tail domain are taken together as a single domain.
• In an embodiment, a gRNA comprises a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 20 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In another embodiment, a gRNA comprises a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 25 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In another embodiment, a gRNA comprises a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 30 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In another embodiment, a gRNA comprises a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 40 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• A cleavage event, e.g., a double strand or single strand break, is generated by a Cas9 molecule. The Cas9 molecule may be an enzymatically active Cas9 (eaCas9) molecule, e.g., an eaCas9 molecule that forms a double strand break in a target nucleic acid or an eaCas9 molecule forms a single strand break in a target nucleic acid (e.g., a nickase molecule).
• In an embodiment, the eaCas9 molecule catalyzes a double strand break.
• In some embodiments, the eaCas9 molecule comprises HNH-like domain cleavage activity but has no, or no significant, N-terminal RuvC-like domain cleavage activity. In this case, the eaCas9 molecule is an HNH-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at D10, e.g., D10A. In other embodiments, the eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity but has no, or no significant, HNH-like domain cleavage activity. In an embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at H840, e.g., H840A. In an embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at N863, e.g., N863A.
• In an embodiment, a single strand break is formed in the strand of the target nucleic acid to which the targeting domain of said gRNA is complementary. In another embodiment, a single strand break is formed in the strand of the target nucleic acid other than the strand to which the targeting domain of said gRNA is complementary.
• In another aspect, disclosed herein is a nucleic acid, e.g., an isolated or non-naturally occurring nucleic acid, e.g., DNA, that comprises (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a CCR5 target position in the CCR5 gene as disclosed herein.
• In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a CCR5 target position in the CCR5 gene to allow alteration, e.g., alteration associated with NHEJ, of a CCR5 target position in the CCR5 gene.
• In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., a first gRNA molecule, comprising a targeting domain configured to target an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain or chromatin modifying protein), sufficiently close to a CCR5 knockdown target position to reduce, decrease or repress expression of the CCR5 gene.
• In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any one of Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18. In an embodiment, the nucleic acid encodes a gRNA molecule comprising a targeting domain is selected from those in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18.
• In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any one of Tables 1A-1F, 2A-2C, 3A-3E, or 4A-4C. In an embodiment, the nucleic acid encodes a gRNA molecule comprising a targeting domain is selected from those in Tables 1A-1F, 2A-2C, 3A-3E, or 4A-4C.
• In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., the first gRNA molecule, comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any one of Tables 5A-5C, 6A-6E, or 7A-7C. In an embodiment, the nucleic acid encodes a gRNA molecule comprising a targeting domain is selected from those in Tables 5A-5C, 6A-6E, or 7A-7C.
• In an embodiment, the nucleic acid encodes a modular gRNA, e.g., one or more nucleic acids encode a modular gRNA. In other embodiments, the nucleic acid encodes a chimeric gRNA. The nucleic acid may encode a gRNA, e.g., the first gRNA molecule, comprising a targeting domain comprising 16 nucleotides or more in length. In an embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 16 nucleotides in length. In another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 17 nucleotides in length. In yet another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 18 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 19 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 20 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 21 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 22 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 23 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 24 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 25 nucleotides in length. In still another embodiment, the nucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising a targeting domain that is 26 nucleotides in length. In an embodiment, a nucleic acid encodes a gRNA comprising from 5′ to 3′: a targeting domain (comprising a “core domain”, and optionally a “secondary domain”); a first complementarity domain; a linking domain; a second complementarity domain; a proximal domain; and a tail domain. In an embodiment, the proximal domain and tail domain are taken together as a single domain.
• In an embodiment, a nucleic acid encodes a gRNA e.g., the first gRNA molecule, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 20 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, a nucleic acid encodes a gRNA e.g., the first gRNA molecule, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 25 nucleotides in length; and a targeting equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, a nucleic acid encodes a gRNA e.g., the first gRNA molecule, comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 30 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, a nucleic acid encodes a gRNA comprising e.g., the first gRNA molecule, a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 40 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, a nucleic acid comprises (a) a sequence that encodes a gRNA molecule e.g., the first gRNA molecule, comprising a targeting domain that is complementary with a target domain in the CCR5 gene as disclosed herein, and further comprising (b) a sequence that encodes a Cas9 molecule.
• The Cas9 molecule may be a nickase molecule, an enzymatically active Cas9 (eaCas9) molecule, e.g., an eaCas9 molecule that forms a double strand break in a target nucleic acid and/or an eaCas9 molecule that forms a single strand break in a target nucleic acid. In an embodiment, a single strand break is formed in the strand of the target nucleic acid to which the targeting domain of said gRNA is complementary. In another embodiment, a single strand break is formed in the strand of the target nucleic acid other than the strand to which to which the targeting domain of said gRNA is complementary.
• In an embodiment, the eaCas9 molecule catalyzes a double strand break.
• In an embodiment, the eaCas9 molecule comprises HNH-like domain cleavage activity but has no, or no significant, N-terminal RuvC-like domain cleavage activity. In another embodiment, the said eaCas9 molecule is an HNH-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at D10, e.g., D10A. In another embodiment, the eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity but has no, or no significant, HNH-like domain cleavage activity. In another embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at H840, e.g., H840A. In another embodiment, the eaCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., the eaCas9 molecule comprises a mutation at N863, e.g., N863A.
• A nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CCR5 gene as disclosed herein; (b) a sequence that encodes a Cas9 molecule, e.g., a Cas9 molecule described herein.
• In an embodiment, the Cas9 molecule is an enzymatically active Cas9 (eaCas9) molecule. In an embodiment, the Cas9 molecule is an enzymatically inactive Cas9 (eiCas9) molecule or a modified eiCas9 molecule, e.g., the eiCas9 molecule is fused to Krüppel-associated box (KRAB) to generate an eiCas9-KRAB fusion protein molecule.
• A nucleic acid disclosed herein may comprise (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CCR5 gene as disclosed herein; (b) a sequence that encodes a Cas9 molecule; and further may comprise (c)(i) a sequence that encodes a second gRNA molecule described herein having a targeting domain that is complementary to a second target domain of the CCR5 gene, and optionally, (c)(ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain of the CCR5 gene; and optionally, (c)(iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain of the CCR5 gene.
• In an embodiment, a nucleic acid encodes a second gRNA molecule comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a CCR5 target position in the CCR5 gene, to allow alteration, e.g., alteration associated with NHEJ, of a CCR5 target position in the CCR5 gene, either alone or in combination with the break positioned by said first gRNA molecule.
• In an embodiment, a nucleic acid encodes a second gRNA molecule comprising a targeting domain configured to target an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain or chromatin modifying protein), sufficiently close to a CCR5 knockdown target position to reduce, decrease or repress expression of the CCR5 gene.
• In an embodiment, a nucleic acid encodes a third gRNA molecule comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a CCR5 target position in the CCR5 gene to allow alteration, e.g., alteration associated with NHEJ, of a CCR5 target position in the CCR5 gene, either alone or in combination with the break positioned by the first and/or second gRNA molecule.
• In an embodiment, a nucleic acid encodes a third gRNA molecule comprising a targeting domain configured to target an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain or chromatin remodeling protein), sufficiently close to a CCR5 knockdown target position to reduce, decrease or repress expression of the CCR5 gene.
• In an embodiment, a nucleic acid encodes a fourth gRNA molecule comprising a targeting domain configured to provide a cleavage event, e.g., a double strand break or a single strand break, sufficiently close to a CCR5 target position in the CCR5 gene to allow alteration, e.g., alteration associated with NHEJ, of a CCR5 target position in the CCR5 gene, either alone or in combination with the break positioned by the first gRNA molecule, the second gRNA molecule and/or the third gRNA molecule.
• In an embodiment, the nucleic acid encodes a second gRNA molecule. The second gRNA is selected to target the same CCR5 target position as the first gRNA molecule. Optionally, the nucleic acid may encode a third gRNA, and further optionally, the nucleic acid may encode a fourth gRNA molecule. The third gRNA molecule and the fourth gRNA molecule are selected to target the same CCR5 target position as the first and second gRNA molecules.
• In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from one of Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18. In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain selected from those in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18. In an embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from one of Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18. In a further embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain selected from those in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18.
• In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from one of Tables 1A-1F, 2A-2C, 3A-3E, or 4A-4C. In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain selected from those in Tables 1A-1F, 2A-2C, 3A-3E, or 4A-4C. In an embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from one of Tables 1A-1F, 2A-2C, 3A-3E, or 4A-4C. In a further embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain selected from those in Tables 1A-1F, 2A-2C, 3A-3E, or 4A-4C.
• In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from one of Tables 5A-5C, 6A-6E, or 7A-7C. In an embodiment, the nucleic acid encodes a second gRNA molecule comprising a targeting domain selected from those in Tables 5A-5C, 6A-6E, or 7A-7C. In an embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain comprising a sequence that is the same as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from one of Tables 5A-5C, 6A-6E, or 7A-7C. In a further embodiment, when a third or fourth gRNA molecule are present, the third and fourth gRNA molecules may independently comprise a targeting domain selected from those in Tables 5A-5C, 6A-6E, or 7A-7C.
• In an embodiment, the nucleic acid encodes a second gRNA which is a modular gRNA, e.g., wherein one or more nucleic acid molecules encode a modular gRNA. In another embodiment, the nucleic acid encoding a second gRNA is a chimeric gRNA. In yet another embodiment, when a nucleic acid encodes a third or fourth gRNA, the third and fourth gRNA may be a modular gRNA or a chimeric gRNA. When multiple gRNAs are used, any combination of modular or chimeric gRNAs may be used.
• A nucleic acid may encode a second, a third, and/or a fourth gRNA, each independently, comprising a targeting domain comprising 16 nucleotides or more in length. In an embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 16 nucleotides in length. In another embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 17 nucleotides in length. In yet another embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 18 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 19 nucleotides in length. In still other embodiments, the nucleic acid encodes a second gRNA comprising a targeting domain that is 20 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 21 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 22 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 23 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 24 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 25 nucleotides in length. In still another embodiment, the nucleic acid encodes a second gRNA comprising a targeting domain that is 26 nucleotides in length.
• In an embodiment, the targeting domain comprises 16 nucleotides.
• In an embodiment, the targeting domain comprises 17 nucleotides.
• In an embodiment, the targeting domain comprises 18 nucleotides.
• In an embodiment, the targeting domain comprises 19 nucleotides.
• In an embodiment, the targeting domain comprises 20 nucleotides.
• In an embodiment, the targeting domain comprises 21 nucleotides.
• In an embodiment, the targeting domain comprises 22 nucleotides.
• In an embodiment, the targeting domain comprises 23 nucleotides.
• In an embodiment, the targeting domain comprises 24 nucleotides.
• In an embodiment, the targeting domain comprises 25 nucleotides.
• In an embodiment, the targeting domain comprises 26 nucleotides.
• In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA, each independently, comprising from 5′ to 3′: a targeting domain (comprising a “core domain”, and optionally a “secondary domain”); a first complementarity domain; a linking domain; a second complementarity domain; a proximal domain; and a tail domain. In some embodiments, the proximal domain and tail domain are taken together as a single domain.
• In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 20 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 25 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 30 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, a nucleic acid encodes a second, a third, and/or a fourth gRNA comprising a linking domain of no more than 25 nucleotides in length; a proximal and tail domain, that taken together, are at least 40 nucleotides in length; and a targeting domain equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, a nucleic acid encodes (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CCR5 gene as disclosed herein, and (b) a sequence that encodes a Cas9 molecule, e.g., a Cas9 molecule described herein. In an embodiment, (a) and (b) are present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., the same adeno-associated virus (AAV) vector. In an embodiment, the nucleic acid molecule is an AAV vector. Exemplary AAV vectors that may be used in any of the described compositions and methods include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, a modified AAV3 vector, an AAV6 vector, a modified AAV6 vector, an AAV8 vector an AAV9 vector, an AAV.rh10 vector, a modified AAV.rh10 vector, an AAV.rh32/33 vector, a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1 vector.
• In another embodiment, (a) is present on a first nucleic acid molecule, e.g. a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (b) is present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecules may be AAV vectors.
• In another embodiment, a nucleic acid encodes (a) a sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CCR5 gene as disclosed herein, and (b) a sequence that encodes a Cas9 molecule, e.g., a Cas9 molecule described herein; and further comprises (c)(i) a sequence that encodes a second gRNA molecule as described herein and optionally, (c)(ii) a sequence that encodes a third gRNA molecule described herein having a targeting domain that is complementary to a third target domain of the CCR5 gene; and optionally, (c)(iii) a sequence that encodes a fourth gRNA molecule described herein having a targeting domain that is complementary to a fourth target domain of the CCR5 gene. In an embodiment, the nucleic acid comprises (a), (b) and (c)(i). In an embodiment, the nucleic acid comprises (a), (b), (c)(i) and (c)(ii). In an embodiment, the nucleic acid comprises (a), (b), (c)(i), (c)(ii) and (c)(iii). Each of (a) and (c)(i) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., the same adeno-associated virus (AAV) vector. In an embodiment, the nucleic acid molecule is an AAV vector.
• In an embodiment, (a) and (c)(i) are on different vectors. For example, (a) may be present on a first nucleic acid molecule, e.g. a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (c)(i) may be present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. In an embodiment, the first and second nucleic acid molecules are AAV vectors.
• In another embodiment, each of (a), (b), and (c)(i) are present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector. In an alternate embodiment, one of (a), (b), and (c)(i) is encoded on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and a second and third of (a), (b), and (c)(i) is encoded on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.
• In an embodiment, (a) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, a first AAV vector; and (b) and (c)(i) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.
• In another embodiment, (b) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (a) and (c)(i) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.
• In another embodiment, (c)(i) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (b) and (a) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.
• In another embodiment, each of (a), (b) and (c)(i) are present on different nucleic acid molecules, e.g., different vectors, e.g., different viral vectors, e.g., different AAV vector. For example, (a) may be on a first nucleic acid molecule, (b) on a second nucleic acid molecule, and (c)(i) on a third nucleic acid molecule. The first, second and third nucleic acid molecule may be AAV vectors.
• In another embodiment, when a third and/or fourth gRNA molecule are present, each of (a), (b), (c)(i), (c)(ii) and (c)(iii) may be present on the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., an AAV vector. In an embodiment, the nucleic acid molecule is an AAV vector. In an alternate embodiment, each of (a), (b), (c)(i), (c)(ii) and (c)(iii) may be present on the different nucleic acid molecules, e.g., different vectors, e.g., the different viral vectors, e.g., different AAV vectors. In a further embodiment, each of (a), (b), (c)(i), (c)(ii) and (c)(iii) may be present on more than one nucleic acid molecule, but fewer than five nucleic acid molecules, e.g., AAV vectors.
• The nucleic acids described herein may comprise a promoter operably linked to the sequence that encodes the gRNA molecule of (a), e.g., a promoter described herein. The nucleic acid may further comprise a second promoter operably linked to the sequence that encodes the second, third and/or fourth gRNA molecule of (c), e.g., a promoter described herein. The promoter and second promoter differ from one another. In some embodiments, the promoter and second promoter are the same.
• The nucleic acids described herein may further comprise a promoter operably linked to the sequence that encodes the Cas9 molecule of (b), e.g., a promoter described herein.
• In another aspect, disclosed herein is a composition comprising (a) a gRNA molecule comprising a targeting domain that is complementary with a target domain in the CCR5 gene, as described herein. The composition of (a) may further comprise (b) a Cas9 molecule, e.g., a Cas9 molecule as described herein. A composition of (a) and (b) may further comprise (c) a second, third and/or fourth gRNA molecule, e.g., a second, third and/or fourth gRNA molecule described herein. In an embodiment, the composition is a pharmaceutical composition. The compositions described herein, e.g., pharmaceutical compositions described herein, can be used in the treatment or prevention of HIV or AIDS in a subject, e.g., in accordance with a method disclosed herein.
• In another aspect, disclosed herein is a method of altering a cell, e.g., altering the structure, e.g., altering the sequence, of a target nucleic acid of a cell, comprising contacting said cell with: (a) a gRNA that targets the CCR5 gene, e.g., a gRNA as described herein; (b) a Cas9 molecule, e.g., a Cas9 molecule as described herein; and optionally, (c) a second, third and/or fourth gRNA that targets CCR5 gene, e.g., a second, third and/or fourth gRNA as described herein.
• In an embodiment, the method comprises contacting said cell with (a) and (b).
• In an embodiment, the method comprises contacting said cell with (a), (b), and (c).
• The gRNA of (a) and optionally (c) may be selected from any of Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18, or a gRNA that differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any of Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18.
• In an embodiment, the method comprises contacting a cell from a subject suffering from or likely to develop an HIV infection or AIDS. The cell may be from a subject who does not have a mutation at a CCR5 target position.
• In an embodiment, the cell being contacted in the disclosed method is a target cell from a circulating blood cell, a progenitor cell, or a stem cell, e.g., a hematopoietic stem cell (HSC) or a hematopoietic stem/progenitor cell (HSPC). In an embodiment, the target cell is a T cell (e.g., a CD4+ T cell, a CD8+ T cell, a helper T cell, a regulatory T cell, a cytotoxic T cell, a memory T cell, a T cell precursor or a natural killer T cell), a B cell (e.g., a progenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasma B cell), a monocyte, a megakaryocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a reticulocyte, a lymphoid progenitor cell, a myeloid progenitor cell, or a hematopoietic stem cell. In an embodiment, the target cell is a bone marrow cell, (e.g., a lymphoid progenitor cell, a myeloid progenitor cell, an erythroid progenitor cell, a hematopoietic stem cell, or a mesenchymal stem cell). In an embodiment, the cell is a CD4 cell, a T cell, a gut associated lymphatic tissue (GALT), a macrophage, a dendritic cell, a myeloid precursor cell, or a microglia. The contacting may be performed ex vivo and the contacted cell may be returned to the subject's body after the contacting step. In another embodiment, the contacting step may be performed in vivo.
• In an embodiment, the method of altering a cell as described herein comprises acquiring knowledge of the presence of a CCR5 target position in said cell, prior to the contacting step. Acquiring knowledge of the presence of a CCR5 target position in the cell may be by sequencing the CCR5 gene, or a portion of the CCR5 gene.
• In an embodiment, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that expresses at least one of (a), (b), and (c). In an embodiment, the contacting step of the method comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, that encodes each of (a), (b), and (c). In another embodiment, the contacting step of the method comprises delivering to the cell a Cas9 molecule of (b) and a nucleic acid which encodes a gRNA of (a) and optionally, a second gRNA of (c)(i) (and further optionally, a third gRNA of (c)(ii) and/or fourth gRNA of (c)(iii).
• In an embodiment, the contacting step comprises contacting the cell with a nucleic acid, e.g., a vector, e.g., an AAV vector, e.g., an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV.rh10 vector, a modified AAV.rh10 vector, an AAV.rh32/33 vector, a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1 vector. a described herein.
• In an embodiment, the contacting step comprises delivering to the cell a Cas9 molecule of (b), as a protein or an mRNA, and a nucleic acid which encodes a gRNA of (a) and optionally a second, third and/or fourth gRNA of (c).
• In an embodiment, the contacting step comprises delivering to the cell a Cas9 molecule of (b), as a protein or an mRNA, said gRNA of (a), as an RNA, and optionally said second, third and/or fourth gRNA of (c), as an RNA.
• In an embodiment, the contacting step comprises delivering to the cell a gRNA of (a) as an RNA, optionally the second, third and/or fourth gRNA of (c) as an RNA, and a nucleic acid that encodes the Cas9 molecule of (b).
• In an embodiment, the contacting step further comprises contacting the cell with an HSC self-renewal agonist, e.g., UM171 ((1r,4r)-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine) or a pyrimidoindole derivative described in Fares et al., Science, 2014, 345(6203): 1509-1512). In an embodiment, the cell is contacted with the HSC self-reneal agonist before (e.g., at least 1, 2, 4, 8, 12, 24, 36, or 48 hours before, e.g., about 2 hours before) the cell is contacted with a gRNA molecule and/or a Cas9 molecule. In another embodiment, the cell is contacted with the HSC self-reneal agonist after (e.g., at least 1, 2, 4, 8, 12, 24, 36, or 48 hours after, e.g., about 24 hours after) the cell is contacted with a gRNA molecule and/or a Cas9 molecule. In yet another embodiment, the cell is contacted with the HSC self-reneal agonist before (e.g., at least 1, 2, 4, 8, 12, 24, 36, or 48 hours before) and after (e.g., at least 1, 2, 4, 8, 12, 24, 36, or 48 hours after) the cell is contacted with a gRNA molecule and/or a Cas9 molecule. In an embodiment, the cell is contacted with the HSC self-reneal agonist about 2 hours before and about 24 hours after the cell is contacted with a gRNA molecule and/or a Cas9 molecule. In an embodiment, the cell is contacted with the HSC self-reneal agonist at the same time the cell is contacted with a gRNA molecule and/or a Cas9 molecule. In an embodiment, the HSC self-renewal agonist, e.g., UM171, is used at a concentration between 5 and 200 nM, e.g., between 10 and 100 nM or between 20 and 50 nM, e.g., about 40 nM.
• In another aspect, disclosed herein is a cell or a population of cells produced (e.g., altered) by a method described herein.
• In another aspect, disclosed herein is a method of treating a subject suffering from or likely to develop an HIV infection or AIDS, e.g., altering the structure, e.g., sequence, of a target nucleic acid of the subject, comprising contacting the subject (or a cell from the subject) with:
• (a) a gRNA that targets the CCR5 gene, e.g., a gRNA disclosed herein;
• (b) a Cas9 molecule, e.g., a Cas9 molecule disclosed herein; and
• optionally, (c)(i) a second gRNA that targets the CCR5 gene, e.g., a second gRNA disclosed herein, and
• further optionally, (c)(ii) a third gRNA, and still further optionally, (c)(iii) a fourth gRNA that target the CCR5 gene, e.g., a third and fourth gRNA disclosed herein.
• In some embodiments, contacting comprises contacting with (a) and (b).
• In some embodiments, contacting comprises contacting with (a), (b), and (c)(i). In some embodiments, contacting comprises contacting with (a), (b), (c)(i) and (c)(ii). In some embodiments, contacting comprises contacting with (a), (b), (c)(i), (c)(ii) and (c)(iii).
• The gRNA of (a) or (c) (e.g., (c)(i), (c)(ii), or (c)(iii)) may be selected from any of Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18, or a gRNA that differs by no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence from any of Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18.
• In an embodiment, the method comprises acquiring knowledge of the presence or absence of a mutation at a CCR5 target position in said subject.
• In an embodiment, the method comprises acquiring knowledge of the presence or absence of a mutation at a CCR5 target position in said subject by sequencing the CCR5 gene or a portion of the CCR5 gene.
• In an embodiment, the method comprises introducing a mutation at a CCR5 target position.
• In an embodiment, the method comprises introducing a mutation at a CCR5 target position by NHEJ.
• When the method comprises introducing a mutation at a CCR5 target position, e.g., by NHEJ in the coding region or a non-coding region, a Cas9 of (b) and at least one guide RNA (e.g., a guide RNA of (a)) are included in the contacting step.
• In an embodiment, a cell of the subject is contacted ex vivo with (a), (b) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii). In an embodiment, said cell is returned to the subject's body.
• In an embodiment, a cell of the subject is contacted is in vivo with (a), (b) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii). In an embodiment, the cell of the subject is contacted in vivo by intravenous delivery of (a), (b) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
• In an embodiment, the contacting step comprises contacting the subject with a nucleic acid, e.g., a vector, e.g., an AAV vector, described herein, e.g., a nucleic acid that encodes at least one of (a), (b), and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
• In an embodiment, the contacting step comprises delivering to said subject said Cas9 molecule of (b), as a protein or mRNA, and a nucleic acid which encodes (a) and optionally (c)(i), further optionally (c)(ii), and still further optionally (c)(iii).
• In an embodiment, the contacting step comprises delivering to the subject the Cas9 molecule of (b), as a protein or mRNA, said gRNA of (a), as an RNA, and optionally said second gRNA of (c)(i), further optionally said third gRNA of (c)(ii), and still further optionally said fourth gRNA of (c)(iii), as an RNA.
• In an embodiment, the contacting step comprises delivering to the subject the gRNA of (a), as an RNA, optionally said second gRNA of (c)(i), further optionally said third gRNA of (c)(ii), and still further optionally said fourth gRNA of (c)(iii), as an RNA, and a nucleic acid that encodes the Cas9 molecule of (b).
• In another aspect, disclosed herein is a reaction mixture comprising a gRNA molecule, a nucleic acid, or a composition described herein, and a cell, e.g., a cell from a subject having, or likely to develop and HIV infection or AIDS, or a subject having a mutation at a CCR5 target position (e.g., a heterozygous carrier of a CCR5 mutation).
• In another aspect, disclosed herein is a kit comprising, (a) a gRNA molecule described herein, or a nucleic acid that encodes the gRNA, and one or more of the following:
• (b) a Cas9 molecule, e.g., a Cas9 molecule described herein, or a nucleic acid or mRNA that encodes the Cas9;
• (c)(i) a second gRNA molecule, e.g., a second gRNA molecule described herein or a nucleic acid that encodes (c)(i);
• (c)(ii) a third gRNA molecule, e.g., a third gRNA molecule described herein or a nucleic acid that encodes (c)(ii);
• (c)(iii) a fourth gRNA molecule, e.g., a fourth gRNA molecule described herein or a nucleic acid that encodes (c)(iii).
• In an embodiment, the kit comprises a nucleic acid, e.g., an AAV vector, that encodes one or more of (a), (b), (c)(i), (c)(ii), and (c)(iii).
• In yet another aspect, disclosed herein is a gRNA molecule, e.g., a gRNA molecule described herein, for use in treating, or delaying the onset or progression of, HIV infection or
• AIDS in a subject, e.g., in accordance with a method of treating, or delaying the onset or progression of, HIV infection or AIDS as described herein.
• In an embodiment, the gRNA molecule in used in combination with a Cas9 molecule, e.g., a Cas9 molecule described herein. Additionally or alternatively, in an embodiment, the gRNA molecule is used in combination with a second, third and/or fourth gRNA molecule, e.g., a second, third and/or fourth gRNA molecule described herein.
• In still another aspect, disclosed herein is use of a gRNA molecule, e.g., a gRNA molecule described herein, in the manufacture of a medicament for treating, or delaying the onset or progression of, HIV infection or AIDS in a subject, e.g., in accordance with a method of treating, or delaying the onset or progression of, HIV infection or AIDS as described herein.
• In an embodiment, the medicament comprises a Cas9 molecule, e.g., a Cas9 molecule described herein. Additionally or alternatively, in an embodiment, the medicament comprises a second, third and/or fourth gRNA molecule, e.g., a second, third and/or fourth gRNA molecule described herein.
• The gRNA molecules and methods, as disclosed herein, can be used in combination with a governing gRNA molecule. As used herein, a governing gRNA molecule refers to a gRNA molecule comprising a targeting domain which is complementary to a target domain on a nucleic acid that encodes a component of the CRISPR/Cas system introduced into a cell or subject. For example, the methods described herein can further include contacting a cell or subject with a governing gRNA molecule or a nucleic acid encoding a governing molecule. In an embodiment, the governing gRNA molecule targets a nucleic acid that encodes a Cas9 molecule or a nucleic acid that encodes a target gene gRNA molecule. In an embodiment, the governing gRNA comprises a targeting domain that is complementary to a target domain in a sequence that encodes a Cas9 component, e.g., a Cas9 molecule or target gene gRNA molecule. In an embodiment, the target domain is designed with, or has, minimal homology to other nucleic acid sequences in the cell, e.g., to minimize off-target cleavage. For example, the targeting domain on the governing gRNA can be selected to reduce or minimize off-target effects. In an embodiment, a target domain for a governing gRNA can be disposed in the control or coding region of a Cas9 molecule or disposed between a control region and a transcribed region. In an embodiment, a target domain for a governing gRNA can be disposed in the control or coding region of a target gene gRNA molecule or disposed between a control region and a transcribed region for a target gene gRNA. While not wishing to be bound by theory, in an embodiment, it is believed that altering, e.g., inactivating, a nucleic acid that encodes a Cas9 molecule or a nucleic acid that encodes a target gene gRNA molecule can be effected by cleavage of the targeted nucleic acid sequence or by binding of a Cas9 molecule/governing gRNA molecule complex to the targeted nucleic acid sequence.
• The compositions, reaction mixtures and kits, as disclosed herein, can also include a governing gRNA molecule, e.g., a governing gRNA molecule disclosed herein.
• Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
• Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.
• Other features and advantages of the invention will be apparent from the detailed description, drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
• FIGS. 1A-1I are representations of several exemplary gRNAs.
• FIG. 1A depicts a modular gRNA molecule derived in part (or modeled on a sequence in part) from Streptococcus pyogenes (S. pyogenes) as a duplexed structure (SEQ ID NOS: 42 and 43, respectively, in order of appearance);
• FIG. 1B depicts a unimolecular (or chimeric) gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 44);
• FIG. 1C depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 45);
• FIG. 1D depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 46);
• FIG. 1E depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 47);
• FIG. 1F depicts a modular gRNA molecule derived in part from Streptococcus thermophilus (S. thermophilus) as a duplexed structure (SEQ ID NOS: 48 and 49, respectively, in order of appearance);
• FIG. 1G depicts an alignment of modular gRNA molecules of S. pyogenes and S. thermophilus (SEQ ID NOS: 50-53, respectively, in order of appearance).
• FIGS. 1H-1I depicts additional exemplary structures of unimolecular gRNA molecules. FIG. 1H shows an exemplary structure of a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 45). FIG. 1I shows an exemplary structure of a unimolecular gRNA molecule derived in part from S. aureus as a duplexed structure (SEQ ID NO: 40).
• FIGS. 2A-2G depict an alignment of Cas9 sequences from Chylinski et al. (RNA Biol. 2013; 10(5): 726-737). The N-terminal RuvC-like domain is boxed and indicated with a “Y”. The other two RuvC-like domains are boxed and indicated with a “B”. The HNH-like domain is boxed and indicated by a “G”. Sm: S. mutans (SEQ ID NO: 1); Sp: S. pyogenes (SEQ ID NO: 2); St: S. thermophilus (SEQ ID NO: 3); Li: L. innocua (SEQ ID NO: 4). Motif: this is a motif based on the four sequences: residues conserved in all four sequences are indicated by single letter amino acid abbreviation; “*” indicates any amino acid found in the corresponding position of any of the four sequences; and “-” indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, or absent.
• FIGS. 3A-3B show an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et at (SEQ ID NOS: 54-103, respectively, in order of appearance). The last line of FIG. 3B identifies 4 highly conserved residues.
• FIGS. 4A-4B show an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS: 104-177, respectively, in order of appearance). The last line of FIG. 4B identifies 3 highly conserved residues.
• FIGS. 5A-5C show an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et at (SEQ ID NOS: 178-252, respectively, in order of appearance). The last line of FIG. 5C identifies conserved residues.
• FIGS. 6A-6B show an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS: 253-302, respectively, in order of appearance). The last line of FIG. 6B identifies 3 highly conserved residues.
• FIGS. 7A-7B depict an alignment of Cas9 sequences from S. pyogenes and Neisseria meningitidis (N. meningitidis). The N-terminal RuvC-like domain is boxed and indicated with a “Y”. The other two RuvC-like domains are boxed and indicated with a “B”. The HNH-like domain is boxed and indicated with a “G”. Sp: S. pyogenes; Nm: N. meningitidis. Motif: this is a motif based on the two sequences: residues conserved in both sequences are indicated by a single amino acid designation; “*” indicates any amino acid found in the corresponding position of any of the two sequences; “-” indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, and “-” indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, or absent.
• FIG. 8 shows a nucleic acid sequence encoding Cas9 of N. meningitidis (SEQ ID NO: 303). Sequence indicated by an “R” is an SV40 NLS; sequence indicated as “G” is an HA tag; and sequence indicated by an “O” is a synthetic NLS sequence; the remaining (unmarked) sequence is the open reading frame (ORF).
• FIGS. 9A-9B are schematic representations of the domain organization of S. pyogenes Cas 9. FIG. 9A shows the organization of the Cas9 domains, including amino acid positions, in reference to the two lobes of Cas9 (recognition (REC) and nuclease (NUC) lobes). FIG. 9B shows the percent homology of each domain across 83 Cas9 orthologs.
• FIG. 10 depicts the efficiency of NHEJ mediated by a Cas9 molecule and exemplary gRNA molecules targeting the CCR5 locus.
• FIG. 11 depicts flow cytometry analysis of genome edited HSCs to determine co-expression of stem cell phenotypic markers CD34 and CD90 and for viability (7-AAD-AnnexinV− cells). CD34+ HSCs maintain phenotype and viability after Nucleofection™ with Cas9 and CCR5 gRNA plasmid DNA (96 hours).
DETAILED DESCRIPTION Definitions
• “CCR5 target position”, as used herein, refers to any position that results in inactivation of the CCR5 gene. In an embodiment, a CCR5 target position refers to any of a CCR5 target knockout position or a CCR5 target knockdown position, as described herein.
• “Domain”, as used herein, is used to describe segments of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.
• Calculations of homology or sequence identity between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
• “Governing gRNA molecule”, as used herein, refers to a gRNA molecule that comprises a targeting domain that is complementary to a target domain on a nucleic acid that comprises a sequence that encodes a component of the CRISPR/Cas system that is introduced into a cell or subject. A governing gRNA does not target an endogenous cell or subject sequence. In an embodiment, a governing gRNA molecule comprises a targeting domain that is complementary with a target sequence on: (a) a nucleic acid that encodes a Cas9 molecule; (b) a nucleic acid that encodes a gRNA which comprises a targeting domain that targets the CCR5 gene (a target gene gRNA); or on more than one nucleic acid that encodes a CRISPR/Cas component, e.g., both (a) and (b). In an embodiment, a nucleic acid molecule that encodes a CRISPR/Cas component, e.g., that encodes a Cas9 molecule or a target gene gRNA, comprises more than one target domain that is complementary with a governing gRNA targeting domain. While not wishing to be bound by theory, in an embodiment, it is believed that a governing gRNA molecule complexes with a Cas9 molecule and results in Cas9 mediated inactivation of the targeted nucleic acid, e.g., by cleavage or by binding to the nucleic acid, and results in cessation or reduction of the production of a CRISPR/Cas system component. In an embodiment, the Cas9 molecule forms two complexes: a complex comprising a Cas9 molecule with a target gene gRNA, which complex will alter the CCR5 gene; and a complex comprising a Cas9 molecule with a governing gRNA molecule, which complex will act to prevent further production of a CRISPR/Cas system component, e.g., a Cas9 molecule or a target gene gRNA molecule. In an embodiment, a governing gRNA molecule/Cas9 molecule complex binds to or promotes cleavage of a control region sequence, e.g., a promoter, operably linked to a sequence that encodes a Cas9 molecule, a sequence that encodes a transcribed region, an exon, or an intron, for the Cas9 molecule. In an embodiment, a governing gRNA molecule/Cas9 molecule complex binds to or promotes cleavage of a control region sequence, e.g., a promoter, operably linked to a gRNA molecule, or a sequence that encodes the gRNA molecule. In an embodiment, the governing gRNA, e.g., a Cas9-targeting governing gRNA molecule, or a target gene gRNA-targeting governing gRNA molecule, limits the effect of the Cas9 molecule/target gene gRNA molecule complex-mediated gene targeting. In an embodiment, a governing gRNA places temporal, level of expression, or other limits, on activity of the Cas9 molecule/target gene gRNA molecule complex. In an embodiment, a governing gRNA reduces off-target or other unwanted activity. In an embodiment, a governing gRNA molecule inhibits, e.g., entirely or substantially entirely inhibits, the production of a component of the Cas9 system and thereby limits, or governs, its activity.
• “Modulator”, as used herein, refers to an entity, e.g., a drug, that can alter the activity (e.g., enzymatic activity, transcriptional activity, or translational activity), amount, distribution, or structure of a subject molecule or genetic sequence. In an embodiment, modulation comprises cleavage, e.g., breaking of a covalent or non-covalent bond, or the forming of a covalent or non-covalent bond, e.g., the attachment of a moiety, to the subject molecule. In an embodiment, a modulator alters the, three dimensional, secondary, tertiary, or quaternary structure, of a subject molecule. A modulator can increase, decrease, initiate, or eliminate a subject activity.
• “Large molecule”, as used herein, refers to a molecule having a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD. Large molecules include proteins, polypeptides, nucleic acids, biologics, and carbohydrates.
• “Polypeptide”, as used herein, refers to a polymer of amino acids having less than 100 amino acid residues. In an embodiment, it has less than 50, 20, or 10 amino acid residues.
• “Reference molecule”, e.g., a reference Cas9 molecule or reference gRNA, as used herein, refers to a molecule to which a subject molecule, e.g., a subject Cas9 molecule of subject gRNA molecule, e.g., a modified or candidate Cas9 molecule is compared. For example, a Cas9 molecule can be characterized as having no more than 10% of the nuclease activity of a reference Cas9 molecule. Examples of reference Cas9 molecules include naturally occurring unmodified Cas9 molecules, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, S. aureus or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology with the Cas9 molecule to which it is being compared. In an embodiment, the reference Cas9 molecule is a sequence, e.g., a naturally occurring or known sequence, which is the parental form on which a change, e.g., a mutation has been made.
• “Replacement”, or “replaced”, as used herein with reference to a modification of a molecule does not require a process limitation but merely indicates that the replacement entity is present.
• “Small molecule”, as used herein, refers to a compound having a molecular weight less than about 2 kD, e.g., less than about 2 kD, less than about 1.5 kD, less than about 1 kD, or less than about 0.75 kD.
• “Subject”, as used herein, may mean either a human or non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). In an embodiment, the subject is a human. In other embodiments, the subject is poultry.
• “Treat”, “treating” and “treatment”, as used herein, mean the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) relieving the disease, i.e., causing regression of the disease state; and (c) curing the disease.
• “Prevent”, “preventing” and “prevention”, as used herein, means the prevention of a disease in a mammal, e.g., in a human, including (a) avoiding or precluding the disease; (2) affecting the predisposition toward the disease, e.g., preventing at least one symptom of the disease or to delay onset of at least one symptom of the disease.
• “X” as used herein in the context of an amino acid sequence, refers to any amino acid (e.g., any of the twenty natural amino acids) unless otherwise specified.
Human Immunodeficiency Virus
• Human Immunodeficiency Virus (HIV) is a virus that causes severe immunodeficiency. In the United States, more than 1 million people are infected with the virus. Worldwide, approximately 30-40 million people are infected.
• HIV is a single-stranded RNA virus that preferentially infects CD4 cells. The virus binds to receptors on the surface of CD4+ cells to enter and infect these cells. This binding and infection step is vital to the pathogenesis of HIV. The virus attaches to the CD4 receptor on the cell surface via its own surface glycoproteins, gp120 and gp41. These proteins are made from the cleavage product of gp160. Gp120 binds to a CD4 receptor and must also bind to another coreceptor in order for the virus to enter the host cell. In macrophage-(M-tropic) viruses, the coreceptor is CCR5 occasionally referred to as the CCR5 receptor. M-tropic virus is found most commonly in the early stages of HIV infection.
• There are two types of HIV—HIV-1 and HIV-2. HIV-1 is the predominant global form and is a more virulent strain of the virus. HIV-2 has lower rates of infection and, at present, predominantly affects populations in West Africa. HIV is transmitted primarily through sexual exposure, although the sharing of needles in intravenous drug use is another mode of transmission.
• As HIV infection progresses, the virus infects CD4 cells and a subject's CD4 counts fall. With declining CD4 counts, a subject is subject to increasing risk of opportunistic infections (OI). Severely declining CD4 counts are associated with a very high likelihood of OIs, specific cancers (such as Kaposi's sarcoma, Burkitt's lymphoma) and wasting syndrome. Normal CD4 counts are between 600-1200 cells/microliter.
• Untreated HIV infection is a chronic, progressive disease that leads to acquired immunodeficiency syndrome (AIDS) and death in the vast majority of subjects. Diagnosis of AIDS is made based on infection with a variety of opportunistic pathogens, presence of certain cancers and/or CD4 counts below 200 cells/μL.
• HIV was untreatable and invariably led to death until the late 1980's. Since then, antiretroviral therapy (ART) has dramatically slowed the course of HIV infection. Highly active antiretroviral therapy (HAART) is the use of three or more agents in combination to slow HIV. Antiretroviral therapy (ART) is indicated in a subject whose CD4 counts has dropped below 500 cells/μL. Viral load is the most common measurement of the efficacy of HIV treatment and disease progression. Viral load measures the amount of HIV RNA present in the blood.
• Treatment with HAART has significantly altered the life expectancy of those infected with HIV. A subject in the developed world who maintains their HAART regimen can expect to live into their 60's and possibly 70's. However, HAART regimens are associated with significant, long term side effects. First, the dosing regimens are complex and associated with strict food requirements. Compliance rates with dosing can be lower than 50% in some populations in the United States. In addition, there are significant toxicities associated with HAART treatment, including diabetes, nausea, malaise, sleep disturbances. A subject who does not adhere to dosing requirements of HAART therapy may have return of viral load in their blood and are at risk for progression to disease and its associated complications.
Methods to Treat or Prevent HIV Infection or AIDS
• Methods and compositions described herein provide for a therapy, e.g., a one-time therapy, or a multi-dose therapy, that prevents or treats HIV infection and/or AIDS. In an embodiment, a disclosed therapy prevents, inhibits, or reduces the entry of HIV into CD4 cells of a subject who is already infected. While not wishing to be bound by theory, in an embodiment, it is believed that knocking out CCR5 on CD4 cells, renders the HIV virus unable to enter CD4 cells. Viral entry into CD4 cells requires interaction of the viral glycoproteins gp41 and gp120 with both the CD4 receptor and acoreceptor, e.g., CCR5. Once a functional coreceptor such as CCR5 has been eliminated from the surface of the CD4 cells, the virus is prevented from binding and entering the host CD4 cells. In an embodiment, the disease does not progress or has delayed progression compared to a subject who has not received the therapy.
• While not wishing to be bound by theory, subjects with naturally occurring CCR5 receptor mutations who have delayed HIV progression may confer protection by the mechanism of action described herein. Subjects with a specific deletion in the CCR5 gene (e.g., the delta 32 deletion) have been shown to have much higher likelihood of being long-term non-progressors (meaning they did not require HAART and their HIV infection did not progress). See, e.g., Stewart G J et al., 1997 The Australian Long-Term Non-Progressor Study Group. Aids. 11:1833-1838. In addition, a subject who was CCR5+ (had a wild type CCR5 receptor) and infected with HIV underwent a bone marrow transplant for acute myeloid lymphoma. See, e.g., Hutter G et al., 2009N ENGL J MED. 360:692-698. The bone marrow transplant (BMT) was from a subject homozygous for a CCR5 delta 32 deletion. Following BMT, the subject did not have progression of HIV and did not require treatment with ART. These subjects offer evidence for the fact that introduction of a protective mutation of the CCR5 gene, or knockout or knockdown of the CCR5 gene prevents, delays or diminishes the ability of HIV to infect the subject. Mutation or deletion of the CCR5 gene, or reduced CCR5 gene expression, should therefore reduce the progression, virulence and pathology of HIV. In an embodiment, a method described herein is used to treat a subject having HIV.
• In an embodiment, a method described herein is used to treat a subject having AIDS.
• In an embodiment, a method described herein is used to prevent, or delay the onset or progression of, HIV infection and AIDS in a subject at high risk for HIV infection.
• In an embodiment, a method described herein results in a selective advantage to survival of treated CD4 cells. Some proportion of CD4 cells will be modified and have a CCR5 protective mutation. These cells are not subject to infection with HIV. Cells that are not modified may be infected with HIV and are expected to undergo cell death. In an embodiment, after the treatment described herein, treated cells survive, while untreated cells die. This selective advantage drives eventual colonization in all body compartments with 100% CCR5-negative CD4 cells derived from treated cells, conferring complete protection in treated subjects against infection with M tropic HIV.
• In an embodiment, the method comprises initiating treatment of a subject prior to disease onset.
• In an embodiment, the method comprises initiating treatment of a subject after disease onset.
• In an embodiment, the method comprises initiating treatment of a subject after disease onset, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 24, 36, 48 or more months after onset of HIV infection or AIDS. While not wishing to be bound by theory, it is believed that this may be effective as disease progression is slow in some cases and a subject may present well into the course of illness.
• In an embodiment, the method comprises initiating treatment of a subject in an advanced stage of disease, e.g., to slow viral replication and viral load.
• Overall, initiation of treatment for a subject at all stages of disease is expected to prevent or reduce disease progression and benefit a subject.
• In an embodiment, the method comprises initiating treatment of a subject prior to disease onset and prior to infection with HIV.
• In an embodiment, the method comprises initiating treatment of a subject in an early stage of disease, e.g., when a subject has tested positive for HIV infection but has no signs or symptoms associated with HIV.
• In an embodiment, the method comprises initiating treatment of a patient at the appearance of a reduced CD4 count or a positive HIV test.
• In an embodiment, the method comprises treating a subject considered at risk for developing HIV infection.
• In an embodiment, the method comprises treating a subject who is the spouse, partner, sexual partner, newborn, infant, or child of a subject with HIV.
• In an embodiment, the method comprises treating a subject for the prevention or reduction of HIV infection.
• In an embodiment, the method comprises treating a subject at the appearance of any of the following findings consistent with HIV: low CD4 count; opportunistic infections associated with HIV, including but not limited to: candidiasis, mycobacterium tuberculosis, cryptococcosis, cryptosporidiosis, cytomegalovirus; and/or malignancy associated with HIV, including but not limited to: lymphoma, Burkitt's lymphoma, or Kaposi's sarcoma.
• In an embodiment, a cell is treated ex vivo and returned to a patient.
• In an embodiment, an autologous CD4 cell can be treated ex vivo and returned to the subject.
• In an embodiment, a heterologous CD4 cells can be treated ex vivo and transplanted into the subject.
• In an embodiment, an autologous stem cell can be treated ex vivo and returned to the subject.
• In an embodiment, a heterologous stem cell can be treated ex vivo and transplanted into the subject.
• In an embodiment, the treatment comprises delivery of gRNA by intravenous injection, intramuscular injection; subcutaneous injection; intrathecal injection; or intraventricular injection.
• In an embodiment, the treatment comprises delivery of a gRNA by an AAV.
• In an embodiment, the treatment comprises delivery of a gRNA by a lentivirus.
• In an embodiment, the treatment comprises delivery of a gRNA by a nanoparticle.
• In an embodiment, the treatment comprises delivery of a gRNA by a parvovirus, e.g., a specifically a modified parvovirus designed to target bone marrow cells and/or CD4 cells.
• In an embodiment, the treatment is initiated after a subject is determined to not have a mutation (e.g., an inactivating mutation, e.g., an inactivating mutation in either or both alleles) in CCR5 by genetic screening, e.g., genotyping, wherein the genetic testing was performed prior to or after disease onset.
Methods of Targeting CCR5
• As disclosed herein, the CCR5 gene can be targeted (e.g., altered) by gene editing, e.g., using CRISPR-Cas9 mediated methods as described herein.
• Methods and compositions discussed herein, provide for targeting (e.g., altering) a CCR5 target position in the CCR5 gene. A CCR5 target position can be targeted (e.g., altered) by gene editing, e.g., using CRISPR-Cas9 mediated methods to target (e.g. alter) the CCR5 gene.
• Disclosed herein are methods for targeting (e.g., altering) a CCR5 target position in the CCR5 gene. Targeting (e.g., altering) the CCR5 target position is achieved, e.g., by:
• (1) knocking out the CCR5 gene:
• (a) insertion or deletion (e.g., NHEJ-mediated insertion or deletion) of one or more nucleotides in close proximity to or within the early coding region of the CCR5 gene, or
• (b) deletion (e.g., NHEJ-mediated deletion) of a genomic sequence including at least a portion of the CCR5 gene, or
• (2) knocking down the CCR5 gene mediated by enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9-fusion protein by targeting non-coding region, e.g., a promoter region, of the gene.
• All approaches give rise to targeting (e.g., alteration) of the CCR5 gene.
• In one embodiment, methods described herein introduce one or more breaks near the early coding region in at least one allele of the CCR5 gene. In another embodiment, methods described herein introduce two or more breaks to flank at least a portion of the CCR5 gene. The two or more breaks remove (e.g., delete) a genomic sequence including at least a portion of the CCR5 gene. In another embodiment, methods described herein comprise knocking down the CCR5 gene mediated by enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9-fusion protein by targeting the promoter region of CCR5 target knockdown position. All methods described herein result in targeting (e.g., alteration) of the CCR5 gene.
• The targeting (e.g., alteration) of the CCR5 gene can be mediated by any mechanism. Exemplary mechanisms that can be associated with the alteration of the CCR5 gene include, but are not limited to, non-homologous end joining (e.g., classical or alternative), microhomology-mediated end joining (MMEJ), homology-directed repair (e.g., endogenous donor template mediated), SDSA (synthesis dependent strand annealing), single strand annealing or single strand invasion.
Knocking Out CCR5 by Introducing an Indel or a Deletion in the CCR5 Gene
• In an embodiment, the method comprises introducing an insertion or deletion of one more nucleotides in close proximity to the CCR5 target knockout position (e.g., the early coding region) of the CCR5 gene. As described herein, in one embodiment, the method comprises the introduction of one or more breaks (e.g., single strand breaks or double strand breaks) sufficiently close to (e.g., either 5′ or 3′ to) the early coding region of the CCR5 target knockout position, such that the break-induced indel could be reasonably expected to span the CCR5 target knockout position (e.g., the early coding region). While not wishing to be bound by theory, it is believed that NHEJ-mediated repair of the break(s) allows for the NHEJ-mediated introduction of an indel in close proximity to within the early coding region of the CCR5 target knockout position.
• In an embodiment, the method comprises introducing a deletion of a genomic sequence comprising at least a portion of the CCR5 gene. As described herein, in an embodiment, the method comprises the introduction of two double stand breaks—one 5′ and the other 3′ to (i.e., flanking) the CCR5 target position. In an embodiment, two gRNAs, e.g., unimolecular (or chimeric) or modular gRNA molecules, are configured to position the two double strand breaks on opposite sides of the CCR5 target knockout position in the CCR5 gene.
• In an embodiment, a single strand break is introduced (e.g., positioned by one gRNA molecule) at or in close proximity to a CCR5 target position in the CCR5 gene. In an embodiment, a single gRNA molecule (e.g., with a Cas9 nickase) is used to create a single strand break at or in close proximity to the CCR5 target position, e.g., the gRNA is configured such that the single strand break is positioned either upstream (e.g., within 500 bp upstream, e.g., within 200 bp upstream) or downstream (e.g., within 500 bp downstream, e.g., within 200 bp downstream) of the CCR5 target position. In an embodiment, the break is positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
• In an embodiment, a double strand break is introduced (e.g., positioned by one gRNA molecule) at or in close proximity to a CCR5 target position in the CCR5 gene. In an embodiment, a single gRNA molecule (e.g., with a Cas9 nuclease other than a Cas9 nickase) is used to create a double strand break at or in close proximity to the CCR5 target position, e.g., the gRNA molecule is configured such that the double strand break is positioned either upstream (e.g., within 500 bp upstream, e.g., within 200 bp upstream) or downstream of (e.g., within 500 bp downstream, e.g., within 200 bp downstream) of a CCR5 target position. In an embodiment, the break is positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
• In an embodiment, two single strand breaks are introduced (e.g., positioned by two gRNA molecules) at or in close proximity to a CCR5 target position in the CCR5 gene. In an embodiment, two gRNA molecules (e.g., with one or two Cas9 nickases) are used to create two single strand breaks at or in close proximity to the CCR5 target position, e.g., the gRNAs molecules are configured such that both of the single strand breaks are positioned e.g., within 500 by upstream, e.g., within 200 bp upstream) or downstream (e.g., within 500 bp downstream, e.g., within 200 bp downstream) of the CCR5 target position. In another embodiment, two gRNA molecules (e.g., with two Cas9 nickases) are used to create two single strand breaks at or in close proximity to the CCR5 target position, e.g., the gRNAs molecules are configured such that one single strand break is positioned upstream (e.g., within 200 bp upstream) and a second single strand break is positioned downstream (e.g., within 200 bp downstream) of the CCR5 target position. In an embodiment, the breaks are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
• In an embodiment, two double strand breaks are introduced (e.g., positioned by two gRNA molecules) at or in close proximity to a CCR5 target position in the CCR5 gene. In an embodiment, two gRNA molecules (e.g., with one or two Cas9 nucleases that are not Cas9 nickases) are used to create two double strand breaks to flank a CCR5 target position, e.g., the gRNA molecules are configured such that one double strand break is positioned upstream (e.g., within 500 bp upstream, e.g., within 200 bp upstream) and a second double strand break is positioned downstream (e.g., within 500 bp downstream, e.g., within 200 bp downstream) of the CCR5 target position. In an embodiment, the breaks are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
• In an embodiment, one double strand break and two single strand breaks are introduced (e.g., positioned by three gRNA molecules) at or in close proximity to a CCR5 target position in the CCR5 gene. In an embodiment, three gRNA molecules (e.g., with a Cas9 nuclease other than a Cas9 nickase and one or two Cas9 nickases) to create one double strand break and two single strand breaks to flank a CCR5 target position, e.g., the gRNA molecules are configured such that the double strand break is positioned upstream or downstream of (e.g., within 500 bp, e.g., within 200 bp upstream or downstream) of the CCR5 target position, and the two single strand breaks are positioned at the opposite site, e.g., downstream or upstream (e.g., within 500 bp, e.g., within 200 bp downstream or upstream), of the CCR5 target position. In an embodiment, the breaks are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
• In an embodiment, four single strand breaks are introduced (e.g., positioned by four gRNA molecules) at or in close proximity to a CCR5 target position in the CCR5 gene. In an embodiment, four gRNA molecule (e.g., with one or more Cas9 nickases are used to create four single strand breaks to flank a CCR5 target position in the CCR5 gene, e.g., the gRNA molecules are configured such that a first and second single strand breaks are positioned upstream (e.g., within 500 bp upstream, e.g., within 200 bp upstream) of the CCR5 target position, and a third and a fourth single stranded breaks are positioned downstream (e.g., within 500 bp downstream, e.g., within 200 bp downstream) of the CCR5 target position. In an embodiment, the breaks are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
• In an embodiment, two or more (e.g., three or four) gRNA molecules are used with one Cas9 molecule. In another embodiment, when two ore more (e.g., three or four) gRNAs are used with two or more Cas9 molecules, at least one Cas9 molecule is from a different species than the other Cas9 molecule(s). For example, when two gRNA molecules are used with two Cas9 molecules, one Cas9 molecule can be from one species and the other Cas9 molecule can be from a different species. Both Cas9 species are used to generate a single or double-strand break, as desired.
Knocking Out CCR5 bp Deleting (e.g., NHEJ-Mediated Deletion) a Genomic Sequence Including at Least a Portion of the CCR5 Gene
• In an embodiment, the method comprises deleting (e.g., NHEJ-mediated deletion) a genomic sequence including at least a portion of the CCR5 gene. As described herein, in one embodiment, the method comprises the introduction two sets of breaks (e.g., a pair of double strand breaks, one double strand break or a pair of single strand breaks, or two pairs of single strand breaks) to flank a region of the CCR5 gene (e.g., a coding region, e.g., an early coding region, or a non-coding region, e.g., a non-coding sequence of the CCR5 gene, e.g., a promoter, an enhancer, an intron, a 3′UTR, and/or a polyadenylation signal). While not wishing to be bound by theory, it is believed that NHEJ-mediated repair of the break(s) allows for alteration of the CCR5 gene as described herein, which reduces or eliminates expression of the gene, e.g., to knock out one or both alleles of the CCR5 gene.
• In an embodiment, two double strand breaks are introduced (e.g., positioned by two gRNA molecules) at or in close proximity to a CCR5 target position in the CCR5 gene. In an embodiment, two gRNA molecules (e.g., with one or two Cas9 nucleases that are not Cas9 nickases) are used to create two double strand breaks to flank a CCR5 target position, e.g., the gRNA molecules are configured such that one double strand break is positioned upstream (e.g., within 500 bp upstream, e.g., within 200 bp upstream) and a second double strand break is positioned downstream (e.g., within 500 bp downstream, e.g., within 200 bp downstream) of the CCR5 target position. In an embodiment, the breaks are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
• In an embodiment, one double strand break and two single strand breaks are introduced (e.g., positioned by three gRNA molecules) at or in close proximity to a CCR5 target position in the CCR5 gene. In an embodiment, three gRNA molecules (e.g., with a Cas9 nuclease other than a Cas9 nickase and one or two Cas9 nickases) to create one double strand break and two single strand breaks to flank a CCR5 target position, e.g., the gRNA molecules are configured such that the double strand break is positioned upstream or downstream of (e.g., within 500 bp, e.g., within 200 bp upstream or downstream) of the CCR5 target position, and the two single strand breaks are positioned at the opposite site, e.g., downstream or upstream (e.g., within 500 bp, e.g., within 200 bp downstream or upstream), of the CCR5 target position. In an embodiment, the breaks are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
• In an embodiment, four single strand breaks are introduced (e.g., positioned by four gRNA molecules) at or in close proximity to a CCR5 target position in the CCR5 gene. In an embodiment, four gRNA molecule (e.g., with one or more Cas9 nickases are used to create four single strand breaks to flank a CCR5 target position in the CCR5 gene, e.g., the gRNA molecules are configured such that a first and second single strand breaks are positioned upstream (e.g., within 500 bp upstream, e.g., within 200 bp upstream) of the CCR5 target position, and a third and a fourth single stranded breaks are positioned downstream (e.g., within 500 bp downstream, e.g., within 200 bp downstream) of the CCR5 target position. In an embodiment, the breaks are positioned to avoid unwanted target chromosome elements, such as repeat elements, e.g., an Alu repeat.
• In an embodiment, two or more (e.g., three or four) gRNA molecules are used with one Cas9 molecule. In another embodiment, when two ore more (e.g., three or four) gRNAs are used with two or more Cas9 molecules, at least one Cas9 molecule is from a different species than the other Cas9 molecule(s). For example, when two gRNA molecules are used with two Cas9 molecules, one Cas9 molecule can be from one species and the other Cas9 molecule can be from a different species. Both Cas9 species are used to generate a single or double-strand break, as desired.
• Knocking Down CCR5 Mediated by an Enzymatically Inactive Cas9 (eiCas9) Molecule
• A targeted knockdown approach reduces or eliminates expression of functional CCR5 gene product. As described herein, in an embodiment, a targeted knockdown is mediated by targeting an enzymatically inactive Cas9 (eiCas9) molecule or an eiCas9 fused to a transcription repressor domain or chromatin modifying protein to alter transcription, e.g., to block, reduce, or decrease transcription, of the CCR5 gene.
• Methods and compositions discussed herein may be used to alter the expression of the CCR5 gene to treat or prevent HIV infection or AIDS by targeting a promoter region of the CCR5 gene. In an embodiment, the promoter region is targeted to knock down expression of the CCR5 gene. A targeted knockdown approach reduces or eliminates expression of functional CCR5 gene product. As described herein, in an embodiment, a targeted knockdown is mediated by targeting an enzymatically inactive Cas9 (eiCas9) or an eiCas9 fused to a transcription repressor domain or chromatin modifying protein to alter transcription, e.g., to block, reduce, or decrease transcription, of the CCR5 gene.
• In an embodiment, one or more eiCas9s may be used to block binding of one or more endogenous transcription factors. In another embodiment, an eiCas9 can be fused to a chromatin modifying protein. Altering chromatin status can result in decreased expression of the target gene. One or more eiCas9s fused to one or more chromatin modifying proteins may be used to alter chromatin status.
• I. gRNA Molecules
• A gRNA molecule, as that term is used herein, refers to a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid. gRNA molecules can be unimolecular (having a single RNA molecule), sometimes referred to herein as “chimeric” gRNAs, or modular (comprising more than one, and typically two, separate RNA molecules). A gRNA molecule comprises a number of domains. The gRNA molecule domains are described in more detail below.
• Several exemplary gRNA structures, with domains indicated thereon, are provided in FIG. 1. While not wishing to be bound by theory, in an embodiment, with regard to the three dimensional form, or intra- or inter-strand interactions of an active form of a gRNA, regions of high complementarity are sometimes shown as duplexes in FIGS. 1A-1G and other depictions provided herein.
• In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5′ to 3′:
• a targeting domain (which is complementary to a target nucleic acid in the CCR5 gene, e.g., a targeting domain from any of Tables 1A-1F);
• a first complementarity domain;
• a linking domain;
• a second complementarity domain (which is complementary to the first complementarity domain);
• a proximal domain; and
• optionally, a tail domain.
• In an embodiment, a modular gRNA comprises:
• • a first strand comprising, preferably from 5′ to 3′;
    • a targeting domain (which is complementary to a target nucleic acid in the CCR5 gene, e.g., a targeting domain from Tables 1A-1F); and
    • a first complementarity domain; and
  • a second strand, comprising, preferably from 5′ to 3′:
    • optionally, a 5′ extension domain;
    • a second complementarity domain;
    • a proximal domain; and
    • optionally, a tail domain.
• The domains are discussed briefly below:
• The Targeting Domain
• FIGS. 1A-1G provide examples of the placement of targeting domains.
• The targeting domain comprises a nucleotide sequence that is complementary, e.g., at least 80, 85, 90, or 95% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. The targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in an embodiment, it is believed that the complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA molecule/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In an embodiment, the target domain itself comprises in the 5′ to 3′ direction, an optional secondary domain, and a core domain. In an embodiment, the core domain is fully complementary with the target sequence. In an embodiment, the targeting domain is 5 to 50 nucleotides in length. The strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the domain can have a modification, e.g., a modification found in Section VIII herein.
• In an embodiment, the targeting domain is 16 nucleotides in length.
• In an embodiment, the targeting domain is 17 nucleotides in length.
• In an embodiment, the targeting domain is 18 nucleotides in length.
• In an embodiment, the targeting domain is 19 nucleotides in length.
• In an embodiment, the targeting domain is 20 nucleotides in length.
• In an embodiment, the targeting domain is 21 nucleotides in length.
• In an embodiment, the targeting domain is 22 nucleotides in length.
• In an embodiment, the targeting domain is 23 nucleotides in length.
• In an embodiment, the targeting domain is 24 nucleotides in length.
• In an embodiment, the targeting domain is 25 nucleotides in length.
• In an embodiment, the targeting domain is 26 nucleotides in length.
• In an embodiment, the targeting domain comprises 16 nucleotides.
• In an embodiment, the targeting domain comprises 17 nucleotides.
• In an embodiment, the targeting domain comprises 18 nucleotides.
• In an embodiment, the targeting domain comprises 19 nucleotides.
• In an embodiment, the targeting domain comprises 20 nucleotides.
• In an embodiment, the targeting domain comprises 21 nucleotides.
• In an embodiment, the targeting domain comprises 22 nucleotides.
• In an embodiment, the targeting domain comprises 23 nucleotides.
• In an embodiment, the targeting domain comprises 24 nucleotides.
• In an embodiment, the targeting domain comprises 25 nucleotides.
• In an embodiment, the targeting domain comprises 26 nucleotides.
• Targeting domains are discussed in more detail below.
• The First Complementarity Domain
• FIGS. 1A-1G provide examples of first complementarity domains.
• The first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, the first complementarity domain is 5 to 30 nucleotides in length. In an embodiment, the first complementarity domain is 5 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 22 nucleotides in length. In an embodiment, the first complementary domain is 7 to 18 nucleotides in length. In an embodiment, the first complementary domain is 7 to 15 nucleotides in length. In an embodiment, the first complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
• In an embodiment, the first complementarity domain comprises 3 subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, a central subdomain, and a 3′ subdomain. In an embodiment, the 5′ subdomain is 4-9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In an embodiment, the central subdomain is 1, 2, or 3, e.g., 1, nucleotide in length. In an embodiment, the 3′ subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
• The first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a first complementarity domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, first complementarity domain.
• Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section VIII herein.
• First complementarity domains are discussed in more detail below.
• The Linking Domain
• FIGS. 1A-1G provide examples of linking domains.
• A linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently. In an embodiment, the linkage is covalent. In an embodiment, the linking domain covalently couples the first and second complementarity domains, see, e.g., FIGS. 1B-1E. In an embodiment, the linking domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementarity domain. Typically the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
• In modular gRNA molecules the two molecules are associated by virtue of the hybridization of the complementarity domains see e.g., FIG. 1A.
• A wide variety of linking domains are suitable for use in unimolecular gRNA molecules. Linking domains can consist of a covalent bond, or be as short as one or a few nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides in length. In an embodiment, a linking domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In an embodiment, a linking domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides in length. In an embodiment, a linking domain shares homology with, or is derived from, a naturally occurring sequence, e.g., the sequence of a tracrRNA that is 5′ to the second complementarity domain. In an embodiment, the linking domain has at least 50% homology with a linking domain disclosed herein.
• Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section VIII herein.
• Linking domains are discussed in more detail below.
• The 5′ Extension Domain
• In an embodiment, a modular gRNA can comprise additional sequence, 5′ to the second complementarity domain, referred to herein as the 5′ extension domain, see, e.g., FIG. 1A. In an embodiment, the 5′ extension domain is, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides in length. In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.
• The Second Complementarity Domain
• FIGS. 1A-1G provide examples of second complementarity domains.
• The second complementarity domain is complementary with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, e.g., as shown in FIGS. 1A-1B, the second complementarity domain can include sequence that lacks complementarity with the first complementarity domain, e.g., sequence that loops out from the duplexed region.
• In an embodiment, the second complementarity domain is 5 to 27 nucleotides in length. In an embodiment, it is longer than the first complementarity region. In an embodiment the second complementary domain is 7 to 27 nucleotides in length. In an embodiment, the second complementary domain is 7 to 25 nucleotides in length. In an embodiment, the second complementary domain is 7 to 20 nucleotides in length. In an embodiment, the second complementary domain is 7 to 17 nucleotides in length. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
• In an embodiment, the second complementarity domain comprises 3 subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, a central subdomain, and a 3′ subdomain. In an embodiment, the 5′ subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In an embodiment, the central subdomain is 1, 2, 3, 4 or 5, e.g., 3, nucleotides in length. In an embodiment, the 3′ subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.
• In an embodiment, the 5′ subdomain and the 3′ subdomain of the first complementarity domain, are respectively, complementary, e.g., fully complementary, with the 3′ subdomain and the 5′ subdomain of the second complementarity domain.
• The second complementarity domain can share homology with or be derived from a naturally occurring second complementarity domain. In an embodiment, it has at least 50% homology with a second complementarity domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, first complementarity domain.
• Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section VIII herein.
• A Proximal Domain
• FIGS. 1A-1G provide examples of proximal domains.
• In an embodiment, the proximal domain is 5 to 20 nucleotides in length. In an embodiment, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In an embodiment, it has at least 50% homology with a proximal domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, proximal domain.
• Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section VIII herein.
• A Tail Domain
• FIGS. 1A-1G provide examples of tail domains.
• As can be seen by inspection of the tail domains in FIGS. 1A-1E, a broad spectrum of tail domains are suitable for use in gRNA molecules. In an embodiment, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In embodiment, the tail domain nucleotides are from or share homology with sequence from the 5′ end of a naturally occurring tail domain, see e.g., FIG. 1D or FIG. 1E. In an embodiment, the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.
• In an embodiment, the tail domain is absent or is 1 to 50 nucleotides in length. In an embodiment, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In an embodiment, it has at least 50% homology with a tail domain disclosed herein, e.g., an S. pyogenes, S. aureus or S. thermophilus, tail domain.
• In an embodiment, the tail domain includes nucleotides at the 3′ end that are related to the method of in vitro or in vivo transcription. When a T7 promoter is used for in vitro transcription of the gRNA, these nucleotides may be any nucleotides present before the 3′ end of the DNA template. When a U6 promoter is used for in vivo transcription, these nucleotides may be the sequence UUUUUU. When alternate pol-III promoters are used, these nucleotides may be various numbers or uracil bases or may include alternate bases.
• The domains of gRNA molecules are described in more detail below.
• The Targeting Domain
• The “targeting domain” of the gRNA is complementary to the “target domain” on the target nucleic acid. The strand of the target nucleic acid comprising the nucleotide sequence complementary to the core domain of the gRNA is referred to herein as the “complementary strand” of the target nucleic acid. Guidance on the selection of targeting domains can be found, e.g., in Fu Y et al., Nat Biotechnol 2014 (doi: 10.1038/nbt.2808) and Sternberg S H et al., Nature 2014 (doi: 10.1038/nature13011).
• In an embodiment, the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, the targeting domain is 16 nucleotides in length.
• In an embodiment, the targeting domain is 17 nucleotides in length.
• In an embodiment, the targeting domain is 18 nucleotides in length.
• In an embodiment, the targeting domain is 19 nucleotides in length.
• In an embodiment, the targeting domain is 20 nucleotides in length.
• In an embodiment, the targeting domain is 21 nucleotides in length.
• In an embodiment, the targeting domain is 22 nucleotides in length.
• In an embodiment, the targeting domain is 23 nucleotides in length.
• In an embodiment, the targeting domain is 24 nucleotides in length.
• In an embodiment, the targeting domain is 25 nucleotides in length.
• In an embodiment, the targeting domain is 26 nucleotides in length.
• In an embodiment, the targeting domain comprises 16 nucleotides.
• In an embodiment, the targeting domain comprises 17 nucleotides.
• In an embodiment, the targeting domain comprises 18 nucleotides.
• In an embodiment, the targeting domain comprises 19 nucleotides.
• In an embodiment, the targeting domain comprises 20 nucleotides.
• In an embodiment, the targeting domain comprises 21 nucleotides.
• In an embodiment, the targeting domain comprises 22 nucleotides.
• In an embodiment, the targeting domain comprises 23 nucleotides.
• In an embodiment, the targeting domain comprises 24 nucleotides.
• In an embodiment, the targeting domain comprises 25 nucleotides.
• In an embodiment, the targeting domain comprises 26 nucleotides.
• In an embodiment, the targeting domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.
• In an embodiment, the targeting domain is 20+/−5 nucleotides in length.
• In an embodiment, the targeting domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.
• In an embodiment, the targeting domain is 30+/−10 nucleotides in length.
• In an embodiment, the targeting domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In another embodiment, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
• Typically the targeting domain has full complementarity with the target sequence. In an embodiment, the targeting domain has or includes 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain.
• In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5′ end. In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3′ end.
• In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5′ end. In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3′ end.
• In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
• In some embodiments, the targeting domain comprises two consecutive nucleotides that are not complementary to the target domain (“non-complementary nucleotides”), e.g., two consecutive noncomplementary nucleotides that are within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.
• In an embodiment, no two consecutive nucleotides within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain, are not complementary to the targeting domain.
• In an embodiment, there are no noncomplementary nucleotides within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.
• In an embodiment, the targeting domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the targeting domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the targeting domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment, a nucleotide of the targeting domain can comprise a 2′ modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section VIII.
• In some embodiments, the targeting domain includes 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the targeting domain includes 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the targeting domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.
• In some embodiments, the targeting domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.
• In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.
• Modifications in the targeting domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate targeting domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in a system in Section IV. The candidate targeting domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
• In an embodiment, all of the modified nucleotides are complementary to and capable of hybridizing to corresponding nucleotides present in the target domain. In another embodiment, 1, 2, 3, 4, 5, 6, 7 or 8 or more modified nucleotides are not complementary to or capable of hybridizing to corresponding nucleotides present in the target domain.
• In an embodiment, the targeting domain comprises, preferably in the 5′→3′ direction: a secondary domain and a core domain. These domains are discussed in more detail below.
• The Core Domain and Secondary Domain of the Targeting Domain
• The “core domain” of the targeting domain is complementary to the “core domain target” on the target nucleic acid. In an embodiment, the core domain comprises about 8 to about 13 nucleotides from the 3′ end of the targeting domain (e.g., the most 3′ 8 to 13 nucleotides of the targeting domain).
• In an embodiment, the core domain and targeting domain, are independently, 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 15+/−2, or 16+-2, 17+/−2, or 18+/−2, nucleotides in length.
• In an embodiment, the core domain and targeting domain, are independently 10+/−2 nucleotides in length.
• In an embodiment, the core domain and targeting domain, are independently, 10+/−4 nucleotides in length.
• In an embodiment, the core domain and targeting domain are independently 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, nucleotides in length.
• In an embodiment, the core domain and targeting domain are independently 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20 10 to 20 or 15 to 20 nucleotides in length.
• In an embodiment, the core domain and targeting domain are independently 3 to 15, e.g., 6 to 15, 7 to 14, 7 to 13, 6 to 12, 7 to 12, 7 to 11, 7 to 10, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10 or 8 to 9 nucleotides in length.
• The “core domain” is complementary with the “core domain target” of the target nucleic acid. Typically the core domain has exact complementarity with the core domain target. In some embodiments, the core domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the core domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
• The “secondary domain” of the targeting domain of the gRNA is complementary to the “secondary domain target” of the target nucleic acid.
• In an embodiment, the secondary domain is positioned 5′ to the core domain.
• In an embodiment, the secondary domain is absent or optional.
• In an embodiment, if the targeting domain is 26 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.
• In an embodiment, if the targeting domain is 25 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.
• In an embodiment, if the targeting domain is 24 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 11 to 16 nucleotides in length.
• In an embodiment, if the targeting domain is 23 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 10 to 15 nucleotides in length.
• In an embodiment, if the targeting domain is 22 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 9 to 14 nucleotides in length.
• In an embodiment, if the targeting domain is 21 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 8 to 13 nucleotides in length.
• In an embodiment, if the targeting domain is 20 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 7 to 12 nucleotides in length.
• In an embodiment, if the targeting domain is 19 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 6 to 11 nucleotides in length.
• In an embodiment, if the targeting domain is 18 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 5 to 10 nucleotides in length.
• In an embodiment, if the targeting domain is 17 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 4 to 9 nucleotides in length.
• In an embodiment, if the targeting domain is 16 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 3 to 8 nucleotides in length.
• In an embodiment, the secondary domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides in length.
• The secondary domain is complementary with the secondary domain target. Typically the secondary domain has exact complementarity with the secondary domain target. In some embodiments the secondary domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the secondary domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
• In an embodiment, the core domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the core domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the core domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment a nucleotide of the core domain can comprise a 2′ modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section VIII. Typically, a core domain will contain no more than 1, 2, or 3 modifications.
• Modifications in the core domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate core domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate core domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
• In an embodiment, the secondary domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the secondary domain comprises one or more modifications, e.g., modifications that render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the secondary domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment a nucleotide of the secondary domain can comprise a 2′ modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section VIII. Typically, a secondary domain will contain no more than 1, 2, or 3 modifications.
• Modifications in the secondary domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate secondary domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate secondary domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
• In an embodiment, (1) the degree of complementarity between the core domain and its target, and (2) the degree of complementarity between the secondary domain and its target, may differ. In an embodiment, (1) may be greater than (2). In an embodiment, (1) may be less than (2). In an embodiment, (1) and (2) are the same, e.g., each may be completely complementary with its target.
• In an embodiment, (1) the number of modifications (e.g., modifications from Section VIII) of the nucleotides of the core domain and (2) the number of modification (e.g., modifications from Section VIII) of the nucleotides of the secondary domain, may differ. In an embodiment, (1) may be less than (2). In an embodiment, (1) may be greater than (2). In an embodiment, (1) and (2) may be the same, e.g., each may be free of modifications.
• The First and Second Complementarity Domains
• The first complementarity domain is complementary with the second complementarity domain.
• Typically the first domain does not have exact complementarity with the second complementarity domain target. In some embodiments, the first complementarity domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the second complementarity domain. In an embodiment, 1, 2, 3, 4, 5 or 6, e.g., 3 nucleotides, will not pair in the duplex, and, e.g., form a non-duplexed or looped-out region. In an embodiment, an unpaired, or loop-out, region, e.g., a loop-out of 3 nucleotides, is present on the second complementarity domain. In an embodiment, the unpaired region begins 1, 2, 3, 4, 5, or 6, e.g., 4, nucleotides from the 5′ end of the second complementarity domain.
• In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
• In an embodiment, the first and second complementarity domains are:
• independently, 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 15+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2, or 20+/−2, 21+/−2, 22+/−2, 23+/−2, or 24+/−2 nucleotides in length;
• independently, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, nucleotides in length; or
• independently, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 7 to 18, 9 to 16, or 10 to 14 nucleotides in length.
• In an embodiment, the second complementarity domain is longer than the first complementarity domain, e.g., 2, 3, 4, 5, or 6, e.g., 6, nucleotides longer.
• In an embodiment, the first and second complementary domains, independently, do not comprise modifications, e.g., modifications of the type provided in Section VIII.
• In an embodiment, the first and second complementary domains, independently, comprise one or more modifications, e.g., modifications that the render the domain less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment, a nucleotide of the domain can comprise a 2′ modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section VIII.
• In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the first and second complementary domains, independently, include as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.
• In an embodiment, the first and second complementary domains, independently, include modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no two consecutive nucleotides that are modified, within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no nucleotide that is modified within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain.
• Modifications in a complementarity domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate complementarity domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section IV. The candidate complementarity domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
• In an embodiment, the first complementarity domain has at least 60, 70, 80, 85%, 90% or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference first complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, first complementarity domain, or a first complementarity domain described herein, e.g., from FIGS. 1A-1G.
• In an embodiment, the second complementarity domain has at least 60, 70, 80, 85%, 90%, or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference second complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, second complementarity domain, or a second complementarity domain described herein, e.g., from FIGS. 1A-1G.
• The duplexed region formed by first and second complementarity domains is typically 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 base pairs in length (excluding any looped out or unpaired nucleotides).
• In some embodiments, the first and second complementarity domains, when duplexed, comprise 11 paired nucleotides, for example, in the gRNA sequence (one paired strand underlined, one bolded):

• (SEQ ID NO: 5)

  NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC.

  
  In some embodiments, the first and second complementarity domains, when duplexed, comprise 15 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

• (SEQ ID NO: 27)

  NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAAGCAUAGCAA

  GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG

  GUGC.

  
  In some embodiments the first and second complementarity domains, when duplexed, comprise 16 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

• (SEQ ID NO: 28)

  NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGGAAACAGCAUAGC

  AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG

  UCGGUGC.

  
  In some embodiments the first and second complementarity domains, when duplexed, comprise 21 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):

• (SEQ ID NO: 29)

  NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUGGAAACAAA

  ACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU

  GGCACCGAGUCGGUGC.

  
  In some embodiments, nucleotides are exchanged to remove poly-U tracts, for example in the gRNA sequences (exchanged nucleotides underlined):

• (SEQ ID NO: 30)

  NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAGAAAUAGCAAGUUAAUAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;

  (SEQ ID NO: 31)

  NNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAGAAAUAGCAAGUUUAAAU

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;

  or

  (SEQ ID NO: 32)

  NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAUGCUGUAUUGGAAACAAU

  ACAGCAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU

  GGCACCGAGUCGGUGC.

The 5′ Extension Domain
• In an embodiment, a modular gRNA can comprise additional sequence, 5′ to the second complementarity domain. In an embodiment, the 5′ extension domain is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides in length. In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.
• In an embodiment, the 5′ extension domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the 5′ extension domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the 5′ extension domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment, a nucleotide of the 5′ extension domain can comprise a 2′ modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section VIII.
• In some embodiments, the 5′ extension domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the 5′ extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end, e.g., in a modular gRNA molecule. In an embodiment, the 5′ extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end, e.g., in a modular gRNA molecule.
• In some embodiments, the 5′ extension domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or more than 5 nucleotides away from one or both ends of the 5′ extension domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5′ extension domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5′ extension domain.
• Modifications in the 5′ extension domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate 5′ extension domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate 5′ extension domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
• In an embodiment, the 5′ extension domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference 5′ extension domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, 5′ extension domain, or a 5′ extension domain described herein, e.g., from FIGS. 1A-1G.
The Linking Domain
• In a unimolecular gRNA molecule the linking domain is disposed between the first and second complementarity domains. In a modular gRNA molecule, the two molecules are associated with one another by the complementarity domains.
• In an embodiment, the linking domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.
• In an embodiment, the linking domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.
• In an embodiment, the linking domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In other embodiments, the linking domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
• In an embodiment, the linking domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, or 20 nucleotides in length.
• In and embodiment, the linking domain is a covalent bond.
• In an embodiment, the linking domain comprises a duplexed region, typically adjacent to or within 1, 2, or 3 nucleotides of the 3′ end of the first complementarity domain and/or the 5-end of the second complementarity domain. In an embodiment, the duplexed region can be 20+/−10 base pairs in length. In an embodiment, the duplexed region can be 10+/−5, 15+/−5, 20+/−5, or 30+/−5 base pairs in length. In an embodiment, the duplexed region can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs in length.
• Typically the sequences forming the duplexed region have exact complementarity with one another, though in some embodiments as many as 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides are not complementary with the corresponding nucleotides.
• In an embodiment, the linking domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the linking domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the linking domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment a nucleotide of the linking domain can comprise a 2′ modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section VIII.
• In some embodiments, the linking domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications.
• Modifications in a linking domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate linking domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated a system described in Section IV. A candidate linking domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
• In an embodiment, the linking domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference linking domain, e.g., a linking domain described herein, e.g., from FIGS. 1A-1G.
• The Proximal Domain
• In an embodiment, the proximal domain is 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 14+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2, or 20+/−2 nucleotides in length.
• In an embodiment, the proximal domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, the proximal domain is 5 to 20, 7, to 18, 9 to 16, or 10 to 14 nucleotides in length.
• In an embodiment, the proximal domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the proximal domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the proximal domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment a nucleotide of the proximal domain can comprise a 2′ modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section VIII.
• In some embodiments, the proximal domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the proximal domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end, e.g., in a modular gRNA molecule. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end, e.g., in a modular gRNA molecule.
• In some embodiments, the proximal domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain.
• Modifications in the proximal domain can be selected so as to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate proximal domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section IV. The candidate proximal domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
• In an embodiment, the proximal domain has at least 60, 70, 80, 85 90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference proximal domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, proximal domain, or a proximal domain described herein, e.g., from FIGS. 1A-1G.
• The Tail Domain
• In an embodiment, the tail domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.
• In an embodiment, the tail domain is 20+/−5 nucleotides in length.
• In an embodiment, the tail domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.
• In an embodiment, the tail domain is 25+/−10 nucleotides in length.
• In an embodiment, the tail domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.
• In other embodiments, the tail domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
• In an embodiment, the tail domain is 1 to 20, 1 to 15, 1 to 10, or 1 to 5 nucleotides in length.
• In an embodiment, the tail domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section VIII. However, in an embodiment, the tail domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the tail domain can be modified with a phosphorothioate, or other modification(s) from Section VIII. In an embodiment a nucleotide of the tail domain can comprise a 2′ modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or other modification(s) from Section VIII.
• In some embodiments, the tail domain can have as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.
• In an embodiment, the tail domain comprises a tail duplex domain, which can form a tail duplexed region. In an embodiment, the tail duplexed region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs in length. In an embodiment, a further single stranded domain, exists 3′ to the tail duplexed domain. In an embodiment, this domain is 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment it is 4 to 6 nucleotides in length.
• In an embodiment, the tail domain has at least 60, 70, 80, or 90% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference tail domain, e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus or S. thermophilus, tail domain, or a tail domain described herein, e.g., from FIGS. 1A-1G.
• In an embodiment, the proximal and tail domain, taken together comprise the following sequences:

• (SEQ ID NO: 33)

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU,

  or

  (SEQ ID NO: 34)

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGGUGC,

  or

  (SEQ ID NO: 35)

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAU

  C,

  or

  (SEQ ID NO: 36)

  AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG,

  or

  (SEQ ID NO: 37)

  AAGGCUAGUCCGUUAUCA,

  or

  (SEQ ID NO: 38)

  AAGGCUAGUCCG.

• In an embodiment, the tail domain comprises the 3′ sequence UUUUUU, e.g., if a U6 promoter is used for transcription.
• In an embodiment, the tail domain comprises the 3′ sequence UUUU, e.g., if an H1 promoter is used for transcription.
• In an embodiment, tail domain comprises variable numbers of 3′ Us depending, e.g., on the termination signal of the pol-III promoter used.
• In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template if a T7 promoter is used.
• In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template, e.g., if in vitro transcription is used to generate the RNA molecule.
• In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template, e.g., if a pol-II promoter is used to drive transcription.
• Modifications in the tail domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section IV. gRNAs having a candidate tail domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section IV. The candidate tail domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
• In an embodiment, the tail domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain.
• In an embodiment, a gRNA has the following structure:
• 5′ [targeting domain]-[first complementarity domain]-[linking domain]-[second complementarity domain]-[proximal domain]-[tail domain]-3′
• wherein, the targeting domain comprises a core domain and optionally a secondary domain, and is 10 to 50 nucleotides in length;
• the first complementarity domain is 5 to 25 nucleotides in length and, in an embodiment, has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference first complementarity domain disclosed herein;
• the linking domain is 1 to 5 nucleotides in length;
• the second complementarity domain is 5 to 27 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference second complementarity domain disclosed herein;
• the proximal domain is 5 to 20 nucleotides in length and, in an embodiment, has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference proximal domain disclosed herein; and
• the tail domain is absent or a nucleotide sequence is 1 to 50 nucleotides in length and, in an embodiment, has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference tail domain disclosed herein.
• Exemplary Chimeric gRNAs
• In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5′ to 3′:
• a targeting domain (which is complementary to a target nucleic acid);
• a first complementarity domain, e.g., comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides;
• a linking domain;
• a second complementarity domain (which is complementary to the first complementarity domain);
• a proximal domain; and
• a tail domain, wherein,
• (a) the proximal and tail domain, when taken together, comprise
• at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;
• (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain; or
• (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.
• In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the unimolecular, or chimeric, gRNA molecule (comprising a targeting domain, a first complementary domain, a linking domain, a second complementary domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is depicted as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us, which serve as a termination signal for the U6 promoter, but which could be either absent or fewer in number: NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUU (SEQ ID NO: 45). In an embodiment, the unimolecular, or chimeric, gRNA molecule is a S. pyogenes gRNA molecule.
• In some embodiments, the unimolecular, or chimeric, gRNA molecule (comprising a targeting domain, a first complementary domain, a linking domain, a second complementary domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is depicted as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us, which serve as a termination signal for the U6 promoter, but which could be either absent or fewer in number: NNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGGAAACAGAAUCUACUAAAAC AAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUUU (SEQ ID NO: 40). In an embodiment, the unimolecular, or chimeric, gRNA molecule is a S. aureus gRNA molecule.
• The sequences and structures of exemplary chimeric gRNAs are also shown in FIGS. 1H-1I.
• Exemplary Modular gRNAs
• In an embodiment, a modular gRNA comprises:
• • a first strand comprising, preferably from 5′ to 3′;
    • a targeting domain, e.g., comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides;
    • a first complementarity domain; and
    • a second strand, comprising, preferably from 5′ to 3′:
    • optionally a 5′ extension domain;
    • a second complementarity domain;
    • a proximal domain; and
    • a tail domain,
  • wherein:
• (a) the proximal and tail domain, when taken together, comprise
• at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;
• (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain; or
• (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.
• In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length.
• In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.
• In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.
• In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
• II. Methods for Designing gRNAs
• Methods for designing gRNAs are described herein, including methods for selecting, designing and validating target domains. Exemplary targeting domains are also provided herein. Targeting Domains discussed herein can be incorporated into the gRNAs described herein.
• Methods for selection and validation of target sequences as well as off-target analyses are described, e.g., in Mali et al., 2013 SCIENCE 339(6121): 823-826; Hsu et al. NAT BIOTECHNOL, 31(9): 827-32; Fu et al., 2014 NAT BIOTECHNOL, doi: 10.1038/nbt.2808. PubMed PMID: 24463574; Heigwer et al., 2014 NAT METHODS 11(2):122-3. doi: 10.1038/nmeth.2812. PubMed PMID: 24481216; Bae et al., 2014 BIOINFORMATICS PubMed PMID: 24463181; Xiao A et al., 2014 BIOINFORMATICS PubMed PMID: 24389662.
• For example, a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For each possible gRNA choice using S. pyogenes Cas9, the tool can identify all off-target sequences (preceding either NAG or NGG PAMs) across the genome that contain up to certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA is then ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for CRISPR construction, primer design for the on-target Surveyor assay, and primer design for high-throughput detection and quantification of off-target cleavage via next-gen sequencing, can also be included in the tool. Candidate gRNA molecules can be evaluated by art-known methods or as described in Section IV herein.
• Guide RNAs (gRNAs) for use with S. pyogenes, S. aureus and N. meningitidis Cas9s were identified using a DNA sequence searching algorithm. Guide RNA design was carried out using a custom guide RNA design software based on the public tool cas-offinder (reference: Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases, Bioinformatics. 2014 Feb. 17. Bae S, Park J, Kim J S. PMID:24463181). Said custom guide RNA design software scores guides after calculating their genomewide off-target propensity. Typically matches ranging from perfect matches to 7 mismatches are considered for guides ranging in length from 17 to 24. Once the off-target sites are computationally determined, an aggregate score is calculated for each guide and summarized in a tabular output using a web-interface. In addition to identifying potential gRNA sites adjacent to PAM sequences, the software also identifies all PAM adjacent sequences that differ by 1, 2, 3 or more nucleotides from the selected gRNA sites. Genomic DNA sequence for each gene was obtained from the UCSC Genome browser and sequences were screened for repeat elements using the publically available RepeatMasker program. RepeatMasker searches input DNA sequences for repeated elements and regions of low complexity. The output is a detailed annotation of the repeats present in a given query sequence.
• Following identification, gRNAs were ranked into tiers based on their distance to the target site, their orthogonality or presence of a 5′ G (based on identification of close matches in the human genome containing a relevant PAM, e.g., in the case of S. pyogenes, a NGG PAM, in the case of S. aureus, NNGRR (e.g, a NNGRRT or NNGRRV) PAM, and in the case of N. meningitides, a NNNNGATT or NNNNGCTT PAM. Orthogonality refers to the number of sequences in the human genome that contain a minimum number of mismatches to the target sequence. A “high level of orthogonality” or “good orthogonality” may, for example, refer to 20-mer gRNAs that have no identical sequences in the human genome besides the intended target, nor any sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage.
• As an example, for S. pyogenes and N. meningitides targets, 17-mer, or 20-mer gRNAs were designed. As another example, for S. aureus targets, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer and 24-mer gRNAs were designed. Targeting domains, disclosed herein, may comprise the 17-mer described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C, e.g., the targeting domains of 18 or more nucleotides may comprise the 17-mer gRNAs described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C. Targeting domains, disclosed herein, may comprises the 18-mer described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C, e.g., the targeting domains of 19 or more nucleotides may comprise the 18-mer gRNAs described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C. Targeting domains, disclosed herein, may comprises the 19-mer described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C, e.g., the targeting domains of 20 or more nucleotides may comprise the 19-mer gRNAs described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C. Targeting domains, disclosed herein, may comprises the 20-mer gRNAs described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C e.g., the targeting domains of 21 or more nucleotides may comprise the 20-mer gRNAs described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C. Targeting domains, disclosed herein, may comprises the 21-mer described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C e.g., the targeting domains of 22 or more nucleotides may comprise the 21-mer gRNAs described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C. Targeting domains, disclosed herein, may comprises the 22-mer described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C, e.g., the targeting domains of 23 or more nucleotides may comprise the 22-mer gRNAs described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C. Targeting domains, disclosed herein, may comprises the 23-mer described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C e.g., the targeting domains of 24 or more nucleotides may comprise the 23-mer gRNAs described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C. Targeting domains, disclosed herein, may comprises the 24-mer described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C, e.g., the targeting domains of 25 or more nucleotides may comprise the 24-mer gRNAs described in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E or 7A-7C. gRNAs were identified for both single-gRNA nuclease cleavage and for a dual-gRNA paired “nickase” strategy. Criteria for selecting gRNAs and the determination for which gRNAs can be used for which strategy is based on several considerations:
• gRNA pairs should be oriented on the DNA such that PAMs are facing out and cutting with the D10A Cas9 nickase will result in 5′ overhangs.
• An assumption that cleaving with dual nickase pairs will result in deletion of the entire intervening sequence at a reasonable frequency. However, it will also often result in indel mutations at the site of only one of the gRNAs. Candidate pair members can be tested for how efficiently they remove the entire sequence versus just causing indel mutations at the site of one gRNA.
• The Targeting Domains discussed herein can be incorporated into the gRNAs described herein.
• Strategies to Identify gRNAs for S. pyogenes, S. Aureus, and N. meningitides to Knock Out the CCR5 Gene
• As an example, two strategies were utilized to identify gRNAs for use with S. pyogenes, S. aureus and N. meningitidis Cas9 enzymes.
• In one strategy, gRNAs were designed for use with S. pyogenes Cas9 enzymes (Tables 1A-1D). While it can be desirable to have gRNAs start with a 5′ G, this requirement was relaxed for some gRNAs in tier 1 in order to identify guides in the correct orientation, within a reasonable distance to the mutation and with a high level of orthogonality. In order to find a pair for the dual-nickase strategy it was necessary to either extend the distance from the mutation or remove the requirement for the 5′G. For selection of tier 2 gRNAs, the distance restriction was relaxed in some cases such that a longer sequence was scanned, but the 5′G was required for all gRNAs. Whether or not the distance requirement was relaxed depended on how many sites were found within the original search window. Tier 3 uses the same distance restriction as tier 2, but removes the requirement for a 5′G. Note that tiers are non-inclusive (each gRNA is listed only once). Tier 4 gRNAs were selected based on location in coding sequence of gene.
• As discussed above, gRNAs were identified for single-gRNA nuclease cleavage as well as for a dual-gRNA paired “nickase” strategy, as indicated.
• gRNAs for use with the Neisseria meningitidis and Staphylococcus aureus Cas9s were identified manually by scanning genomic DNA sequence for the presence of PAM sequences. These gRNAs were not separated into tiers, but are provided in single lists for each species (Table 1E for S. aureus and Table 1F for N. meningitides).
• As discussed above, gRNAs were identified for single-gRNA nuclease cleavage as well as for a dual-gRNA paired “nickase” strategy, as indicated.
• In another strategy, gRNAs were designed for use with S. pyogenes, S. aureus and N. meningitidis Cas9 enzymes. The gRNAs were identified and ranked into 3 tiers for S. pyogenes (Tables 2A-2C). The targeting domain to be used with S. pyogenes Cas9 enzymes for tier 1 gRNA molecules were selected based on (1) distance to a target site (e.g., start codon), e.g., within 500 bp (e.g., downstream) of the target site (e.g., start codon) and (2) a high level of orthogonality. The targeting domain to be used with S. pyogenes Cas9 enzymes for tier 2 gRNA molecules were selected based on (1) distance to the target site (e.g., start codon), e.g., within 500 bp (e.g., downstream) of the target site (e.g., start codon). The targeting domain to be used with S. pyogenes Cas9 enzymes for tier 3 gRNA molecules were selected based on distance to the target site (e.g., start codon), e.g., within reminder of the coding sequence, e.g., downstream of the first 500 bp of coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon). The gRNAs were identified and ranked into 5 tiers for S. aureus, when the relevant PAM was NNGRRT or NNGRRV (Tables 3A-3E). The targeting domain to be used with S. aureus Cas9 enzymes for tier 1 gRNA molecules were selected based on (1) distance to the target site (e.g., start codon), e.g., within 500 bp (e.g., downstream) of the target site (e.g., start codon), (2) a high level of orthogonality, and (3) PAM is NNGRRT. The targeting domain to be used with S. aureus Cas9 enzymes for tier 2 gRNA molecules were selected based on (1) distance to the target site (e.g., start codon), e.g., within 500 bp (e.g., downstream) of the target site (e.g., start codon), and (2) PAM is NNGRRT. The targeting domain to be used with S. aureus Cas9 enzymes for tier 3 gRNA molecules were selected based on (1) distance to a the target site (e.g., start codon), e.g., within 500 bp (e.g., downstream) of the target site (e.g., start codon), and (2) PAM is NNGRRV. The targeting domain to be used with S. aureus Cas9 enzymes for tier 4 gRNA molecules were selected based on (1) distance to the target site (e.g., start codon), e.g., within reminder of the coding sequence, e.g., downstream of the first 500 bp of coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon), and (2) PAM is NNGRRT. The targeting domain to be used with S. aureus Cas9 enzymes for tier 5 gRNA molecules were selected based on (1) distance to the target site (e.g., start codon), e.g., within reminder of the coding sequence, e.g., downstream of the first 500 bp of coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon), and (2) PAM is NNGRRV. The gRNAs were identified and ranked into 3 tiers for N. meningitidis (Tables 4A-4C). The targeting domain to be used with N. meningitidis Cas9 enzymes for tier 1 gRNA molecules were selected based on (1) distance to the target site, e.g., within 500 bp (e.g., downstream) of the target site (e.g., start codon) and (2) a high level of orthogonality. The targeting domain to be used with N. meningitidis Cas9 enzymes for tier 2 gRNA molecules were selected based on (1) distance to the target site (e.g., start codon), e.g., within 500 bp (e.g., downstream) of the target site (e.g., start codon). The targeting domain to be used with N. meningitidis Cas9 enzymes for tier 3 gRNA molecules were selected based on distance to the target site (e.g., start codon), e.g., within reminder of the coding sequence, e.g., downstream of the first 500 bp of coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon). Note that tiers are non-inclusive (each gRNA is listed only once for the strategy). In certain instances, no gRNA was identified based on the criteria of the particular tier.
• In an embodiment, when a single gRNA molecule is used to target a Cas9 nickase to create a single strand break in close proximity to the CCR5 target position, e.g., the gRNA is used to target either upstream of (e.g., within 500 bp, e.g., within 200 bp upstream of the CCR5 target position), or downstream of (e.g., within 500 bp, e.g., within 200 bp downstream of the CCR5 target position) in the CCR5 gene.
• In an embodiment, when a single gRNA molecule is used to target a Cas9 nuclease to create a double strand break to in close proximity to the CCR5 target position, e.g., the gRNA is used to target either upstream of (e.g., within 500 bp, e.g., within 200 bp upstream of the CCR5 target position), or downstream of (e.g., within 500 bp, e.g., within 200 bp downstream of the CCR5 target position) in the CCR5 gene.
• In an embodiment, dual targeting is used to create two double strand breaks to in close proximity to the mutation, e.g., the gRNA is used to target either upstream of (e.g., within 500 bp, e.g., within 200 bp upstream of the CCR5 target position), or downstream of (e.g., within 500 bp, e.g., within 200 bp downstream of the CCR5 target position) in the CCR5 gene. In an embodiment, the first and second gRNAs are used to target two Cas9 nucleases to flank, e.g., the first of gRNA is used to target upstream of (e.g., within 500 bp, e.g., within 200 bp upstream of the CCR5 target position), and the second gRNA is used to target downstream of (e.g., within 500 bp, e.g., within 200 bp downstream of the CCR5 target position) in the CCR5 gene.
• In an embodiment, dual targeting is used to create a double strand break and a pair of single strand breaks to delete a genomic sequence including the CCR5 target position. In an embodiment, the first, second and third gRNAs are used to target one Cas9 nuclease and two Cas9 nickases to flank, e.g., the first gRNA that will be used with the Cas9 nuclease is used to target upstream of (e.g., within 500 bp, e.g., within 200 bp upstream of the CCR5 target position) or downstream of (e.g., within 500 bp, e.g., within 200 bp downstream of the CCR5 target position), and the second and third gRNAs that will be used with the Cas9 nickase pair are used to target the opposite side of the mutation (e.g., within 200 bp upstream or downstream of the CCR5 target position) in the CCR5 gene.
• In an embodiment, when four gRNAs (e.g., two pairs) are used to target four Cas9 nickases to create four single strand breaks to delete genomic sequence including the mutation, the first pair and second pair of gRNAs are used to target four Cas9 nickases to flank, e.g., the first pair of gRNAs are used to target upstream of (e.g., within 500 bp, e.g., within 200 bp upstream of the CCR5 target position), and the second pair of gRNAs are used to target downstream of (e.g., within 500 bp, e.g., within 200 bp downstream of the CCR5 target position) in the CCR5 gene.
• Strategies to Identify gRNAs for S. pyogenes, S. Aureus, and N. meningitides to Knock Down the CCR5 Gene
• In yet another strategy, gRNAs were designed for use with S. pyogenes, S. aureus and N. meningitidis Cas9 enzymes. The gRNAs were identified and ranked into 3 tiers for S. pyogenes (Tables 5A-5C). The targeting domain to be used with S. pyogenes Cas9 enzymes for tier 1 gRNA molecules were selected based on (1) distance to a target site (e.g., the transcription start site), e.g., within 500 bp (e.g., upstream or downstream) of the target site (e.g., the transcription start site) and (2) a high level of orthogonality. The targeting domain to be used with S. pyogenes Cas9 enzymes for tier 2 gRNA molecules were selected based on (1) distance to the target site (e.g., the transcription start site), e.g., within 500 bp (e.g., upstream or downstream) of the target site (e.g., the transcription start site). The targeting domain to be used with S. pyogenes Cas9 enzymes for tier 3 gRNA molecules were selected based on distance to the target site (e.g., the transcription start site), e.g., within the additional 500 bp upstream and downstream of the transcription start site (i.e., extending to 1 kb upstream and downstream of the transcription start site. The gRNAs were identified and ranked into 5 tiers for S. aureus, when the relevant PAM was NNGRRT or NNGRRV (Tables 6A-6E). The targeting domain to be used with S. aureus Cas9 enzymes for tier 1 gRNA molecules were selected based on (1) distance to the target site (e.g., the transcription start site), e.g., within 500 bp (e.g., upstream or downstream) of the target site (e.g., the transcription start site), (2) a high level of orthogonality, and (3) PAM is NNGRRT. The targeting domain to be used with S. aureus Cas9 enzymes for tier 2 gRNA molecules were selected based on (1) distance to the target site (e.g., the transcription start site), e.g., within 500 bp (e.g., upstream or downstream) of the target site (e.g., the transcription start site), and (2) PAM is NNGRRT. The targeting domain to be used with S. aureus Cas9 enzymes for tier 3 gRNA molecules were selected based on (1) distance to a target site (e.g., the transcription start site), e.g., within 500 bp (e.g., upstream or downstream) of the target site (e.g., the transcription start site), and (2) PAM is NNGRRV. The targeting domain to be used with S. aureus Cas9 enzymes for tier 4 gRNA molecules were selected based on (1) distance to the target site (e.g., the transcription start site), e.g., within the additional 500 bp upstream and downstream of the transcription start site (i.e., extending to 1 kb upstream and downstream of the transcription start site, and (2) PAM is NNGRRT. The targeting domain to be used with S. aureus Cas9 enzymes for tier 5 gRNA molecules were selected based on (1) distance to the target site (e.g., the transcription start site), e.g., within the additional 500 bp upstream and downstream of the transcription start site (i.e., extending to 1 kb upstream and downstream of the transcription start site, and (2) PAM is NNGRRV. The gRNAs were identified and ranked into 3 tiers for N. meningitidis (Tables 7A-7C). The targeting domain to be used with N. meningitidis Cas9 enzymes for tier 1 gRNA molecules were selected based on (1) distance to a target site (e.g., the transcription start site), e.g., within 500 bp (e.g., upstream or downstream) of the target site (e.g., the transcription start site) and (2) a high level of orthogonality. The targeting domain to be used with N. meningitidis Cas9 enzymes for tier 2 gRNA molecules were selected based on (1) distance to the target site (e.g., the transcription start site), e.g., within 500 bp (e.g., upstream or downstream) of the target site (e.g., the transcription start site). The targeting domain to be used with N. meningitidis Cas9 enzymes for tier 3 gRNA molecules were selected based on distance to the target site (e.g., the transcription start site), e.g., within the additional 500 bp upstream and downstream of the transcription start site (i.e., extending to 1 kb upstream and downstream of the transcription start site. Note that tiers are non-inclusive (each gRNA is listed only once for the strategy). In certain instances, no gRNA was identified based on the criteria of the particular tier.
• Any of the targeting domains in the tables described herein can be used with a Cas9 nickase molecule to generate a single strand break.
• Any of the targeting domains in the tables described herein can be used with a Cas9 nuclease molecule to generate a double strand break.
• In an embodiment, dual targeting (e.g., dual nicking) is used to create two nicks on opposite DNA strands by using S. pyogenes, S. aureus and N. meningitidis Cas9 nickases with two targeting domains that are complementary to opposite DNA strands, e.g., a gRNA comprising any minus strand targeting domain may be paired any gRNA comprising a plus strand targeting domain provided that the two gRNAs are oriented on the DNA such that PAMs face outward and the distance between the 5′ ends of the gRNAs is 0-50 bp.
• When two gRNAs designed for use to target two Cas9 molecules, one Cas9 can be one species, the second Cas9 can be from a different species. Both Cas9 species are used to generate a single or double-strand break, as desired.
Exemplary Targeting Domains
• Table 1A provides exemplary targeting domains for knocking out the CCR5 gene selected according to first tier parameters, and are selected based on the presence of a 5′ G (except for CCR5-51, -52, -60, -63, -64 and -66), close proximity to the start codon and orthogonality in the human genome. In an embodiment, the targeting domain is the exact complement of the target domain. Any of the targeting domains in the table can be used with a Cas9 molecule (e.g., a S. pyogenes Cas9 molecule) that gives double stranded cleavage. Any of the targeting domains in the table can be used with Cas9 single-stranded break nucleases (nickases) (e.g., S. pyogenes Cas9 single-stranded break nucleases). In an embodiment, dual targeting is used to create two nicks. When selecting gRNAs for use in a nickase pair, one gRNA targets a domain in the complementary strand and the second gRNA targets a domain in the non-complementary strand. In an embodiment, two 20-mer guide RNAs are used to target two S. pyogenes Cas9 nucleases or two S. pyogenes Cas9 nickases, e.g., CCR5-63 and CCR5-49, or CCR5-63 and CCR5-41 are used. In an embodiment, two 17-mer guide RNAs are used to target two Cas9 nucleases or two Cas9 nickases, e.g., CCR5-4 and CCR5-3 are used.

• TABLE 1A
  1st Tier

  				SEQ
  gRNA	DNA		Target Site	ID
  Name	Strand	Targeting Domain	Length	NO

  CCR5-66	−	CCUGCCUCCGCUCUACUCAC	20	387
  CCR5-43	−	GCUGCCGCCCAGUGGGACUU	20	388
  CCR5-51	−	ACAAUGUGUCAACUCUUGAC	20	389
  CCR5-58	−	GGUGACAAGUGUGAUCACUU	20	390
  CCR5-60	+	CCAGGUACCUAUCGAUUGUC		20	391
  CCR5-63	+	CUUCACAUUGAUUUUUUGGC		20	392
  CCR5-47	+	GCAGCAUAGUGAGCCCAGAA		20	393
  CCR5-45	+	GGUACCUAUCGAUUGUCAGG		20	394
  CCR5-49	+	GUGAGUAGAGCGGAGGCAGG		20	395
  CCR5-1	−	GCCUCCGCUCUACUCAC	17	396
  CCR5-3	−	GCCGCCCAGUGGGACUU	17	397
  CCR5-52	−	AUGUGUCAACUCUUGAC	17	398
  CCR5-10	−	GACAAUCGAUAGGUACC	17	399
  CCR5-64	+	CACAUUGAUUUUUUGGC		17	400
  CCR5-4	+	GCAUAGUGAGCCCAGAA		17	401
  CCR5-14	+	GGUACCUAUCGAUUGUC		17	402

• Table 1B provides exemplary targeting domains for knocking out the CCR5 gene selected according to the second tier parameters and are selected based on the presence of a 5′ G and close proximity to the start codon. In an embodiment, the targeting domain is the exact complement of the target domain. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that gives double stranded cleavage. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 single-stranded break nucleases (nickases). In an embodiment, dual targeting is used to create two nicks.

• TABLE 1B
  2nd Tier

  			Target
  gRNA	DNA		Site	SEQ
  Name	Strand	Targeting Domain	Length	ID NO

  CCR5-5	+	GAAAAACAGGUCAGAGA		17	403
  CCR5-13	−	GACAAGUGUGAUCACUU	17	404
  CCR5-85	−	GACAAGUGUGAUCACUUGGG	20	405
  CCR5-12	−	GACGGUCACCUUUGGGG	17	406
  CCR5-8	+	GAGCGGAGGCAGGAGGC		17	407
  CCR5-11	−	GCCAGGACGGUCACCUU	17	408
  CCR5-6	+	GCCUUUUGCAGUUUAUC		17	409
  CCR5-59	−	GCUGUGUUUGCGUCUCUCCC	20	410
  CCR5-9	+	GCUUCACAUUGAUUUUU		17	411
  CCR5-48	+	GGACAGUAAGAAGGAAAAAC		20	412
  CCR5-46	+	GGCAGCAUAGUGAGCCCAGA		20	413
  CCR5-41	−	GGUGUUCAUCUUUGGUUUUG	20	414
  CCR5-50	+	GUAGAGCGGAGGCAGGAGGC		20	415
  CCR5-7	+	GUGAGUAGAGCGGAGGC		17	416
  CCR5-42	−	GUGUUCAUCUUUGGUUUUGU	20	417
  CCR5-129	−	GUGUUUGCGUCUCUCCC	17	418
  CCR5-2	−	GUUCAUCUUUGGUUUUG	17	419
  CCR5-79	−	GUUUGCUUUAAAAGCCAGGA	20	420

• Table 1C provides exemplary targeting domains for knocking out the CCR5 gene selected according to the third tier parameters and are selected based on close proximity to the start codon. In an embodiment, the targeting domain is the exact complement of the target domain. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that gives double stranded cleavage. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 single-stranded break nucleases (nickases). In an embodiment, dual targeting is used to create two nicks.

• TABLE 1C
  3rd Tier

  			Target
  gRNA	DNA		Site	SEQ
  Name	Strand	Targeting Domain	Length	ID NO

  CCR5-87	+	AAAACAGGUCAGAGAUGGCC	20	421
  CCR5-80	−	AAAGCCAGGACGGUCACCUU	20	422
  CCR5-130	+	AACACCAGUGAGUAGAG	17	423
  CCR5-88	+	AACACCAGUGAGUAGAGCGG	20	424
  CCR5-81	−	AAGCCAGGACGGUCACCUUU	20	425
  CCR5-89	+	AAGGAAAAACAGGUCAGAGA	20	426
  CCR5-127	−	AAGUGUGAUCACUUGGG	17	427
  CCR5-86	−	AAGUGUGAUCACUUGGGUGG	20	428
  CCR5-90	+	ACACAGCAUGGACGACAGCC	20	429
  CCR5-119	−	ACAGGGCUCUAUUUUAU	17	430
  CCR5-131	+	ACAGGUCAGAGAUGGCC	17	431
  CCR5-132	+	ACAUUGAUUUUUUGGCA	17	432
  CCR5-133	+	ACCAGUGAGUAGAGCGG	17	433
  CCR5-134	+	ACCUAUCGAUUGUCAGG	17	434
  CCR5-115	−	ACUAUGCUGCCGCCCAG	17	435
  CCR5-135	+	ACUUGUCACCACCCCAA	17	436
  CCR5-136	+	AGAAGGGGACAGUAAGA	17	437
  CCR5-137	+	AGAGCGGAGGCAGGAGG	17	438
  CCR5-138	+	AGAUGGCCAGGUUGAGC	17	439
  CCR5-139	+	AGCAUAGUGAGCCCAGA	17	440
  CCR5-82	−	AGCCAGGACGGUCACCUUUG	20	441
  CCR5-65	+	AGUAGAGCGGAGGCAGG	17	442
  CCR5-91	+	AGUAGAGCGGAGGCAGGAGG	20	443
  CCR5-92	+	AUGAACACCAGUGAGUAGAG	20	444
  CCR5-141	+	AUUUCCAAAGUCCCACU	17	445
  CCR5-93	+	AUUUCCAAAGUCCCACUGGG	20	446
  CCR5-76	−	CAAUGUGUCAACUCUUGACA	20	447
  CCR5-94	+	CACACUUGUCACCACCCCAA	20	448
  CCR5-95	+	CACCCCAAAGGUGACCGUCC	20	449
  CCR5-96	+	CAGAGAUGGCCAGGUUGAGC	20	450
  CCR5-97	+	CAGCAUAGUGAGCCCAGAAG	20	451
  CCR5-143	+	CAGCAUGGACGACAGCC	17	452
  CCR5-125	−	CAGGACGGUCACCUUUG	17	453
  CCR5-83	−	CAGGACGGUCACCUUUGGGG	20	454
  CCR5-144	+	CAGUAAGAAGGAAAAAC	17	455
  CCR5-145	+	CAUAGUGAGCCCAGAAG	17	456
  CCR5-107	−	CAUCAAUUAUUAUACAU	17	457
  CCR5-112	−	CAUCUACCUGCUCAACC	17	458
  CCR5-124	−	CCAGGACGGUCACCUUU	17	459
  CCR5-98	+	CCAGUGAGUAGAGCGGAGGC	20	460
  CCR5-146	+	CCCAAAGGUGACCGUCC	17	461
  CCR5-99	+	CCCAGAAGGGGACAGUAAGA	20	462
  CCR5-57	−	CCUGACAAUCGAUAGGUACC	20	463
  CCR5-73	−	CCUUCUUACUGUCCCCUUCU	20	464
  CCR5-116	−	CUAUGCUGCCGCCCAGU	17	465
  CCR5-74	−	CUCACUAUGCUGCCGCCCAG	20	466
  CCR5-78	−	CUGUGUUUGCUUUAAAAGCC	20	467
  CCR5-100	+	CUUUUAAAGCAAACACAGCA	20	468
  CCR5-101	+	UAAUAAUUGAUGUCAUAGAU	20	469
  CCR5-147	+	UAAUUGAUGUCAUAGAU	17	470
  CCR5-68	−	UACUCACUGGUGUUCAUCUU	20	471
  CCR5-148	+	UAUUUCCAAAGUCCCAC	17	472
  CCR5-77	−	UAUUUUAUAGGCUUCUUCUC	20	473
  CCR5-75	−	UCACUAUGCUGCCGCCCAGU	20	474
  CCR5-108	−	UCACUGGUGUUCAUCUU	17	475
  CCR5-62	+	UCAGCCUUUUGCAGUUUAUC	20	476
  CCR5-55	−	UCAUCCUCCUGACAAUCGAU	20	477
  CCR5-70	−	UCAUCCUGAUAAACUGCAAA	20	478
  CCR5-149	+	UCCAAAGUCCCACUGGG	17	479
  CCR5-121	−	UCCUCCUGACAAUCGAU	17	480
  CCR5-111	−	UCCUGAUAAACUGCAAA	17	481
  CCR5-72	−	UCCUUCUUACUGUCCCCUUC	20	482
  CCR5-114	−	UCUUACUGUCCCCUUCU	17	483
  CCR5-126	−	UGACAAGUGUGAUCACU	17	484
  CCR5-67	−	UGACAUCAAUUAUUAUACAU	20	485
  CCR5-71	−	UGACAUCUACCUGCUCAACC	20	486
  CCR5-150	+	UGCAGUUUAUCAGGAUG	17	487
  CCR5-123	−	UGCUUUAAAAGCCAGGA	17	488
  CCR5-84	−	UGGUGACAAGUGUGAUCACU	20	489
  CCR5-69	−	UGGUUUUGUGGGCAACAUGC	20	490
  CCR5-102	+	UGUAUUUCCAAAGUCCCACU	20	491
  CCR5-128	−	UGUGAUCACUUGGGUGG	17	492
  CCR5-118	−	UGUGUCAACUCUUGACA	17	493
  CCR5-122	−	UGUUUGCUUUAAAAGCC	17	494
  CCR5-151	+	UUAAAGCAAACACAGCA	17	495
  CCR5-103	+	UUCACAUUGAUUUUUUGGCA	20	496
  CCR5-109	−	UUCAUCUUUGGUUUUGU	17	497
  CCR5-113	−	UUCUUACUGUCCCCUUC	17	498
  CCR5-53	−	UUGACAGGGCUCUAUUUUAU	20	499
  CCR5-104	+	UUGUAUUUCCAAAGUCCCAC	20	500
  CCR5-120	−	UUUAUAGGCUUCUUCUC	17	501
  CCR5-105	+	UUUGCUUCACAUUGAUUUUU	20	502
  CCR5-106	+	UUUUGCAGUUUAUCAGGAUG	20	503
  CCR5-110	−	UUUUGUGGGCAACAUGC	17	504

• Table 1D provides exemplary targeting domains for knocking out the CCR5 gene selected according to the fourth tier parameters and are selected on location in coding sequence of gene. In an embodiment, the targeting domain is the exact complement of the target domain. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that gives double stranded cleavage. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 single-stranded break nucleases (nickases). In an embodiment, dual targeting is used to create two nicks.

• TABLE 1D
  4th Tier

  			Target
  gRNA	DNA		Site	SEQ
  Name	Strand	Targeting Domain	Length	ID NO

  CCR5-152	−	CAUACAGUCAGUAUCAAUUC	20	505
  CCR5-153	−	GACAUUAAAGAUAGUCAUCU	20	506
  CCR5-154	−	ACAUUAAAGAUAGUCAUCUU	20	507
  CCR5-155	−	CAUUAAAGAUAGUCAUCUUG	20	508
  CCR5-156	−	AAAGAUAGUCAUCUUGGGGC	20	509
  CCR5-157	−	GGUCCUGCCGCUGCUUGUCA	20	510
  CCR5-158	−	UGUCAUGGUCAUCUGCUACU	20	511
  CCR5-159	−	GUCAUGGUCAUCUGCUACUC	20	512
  CCR5-160	−	GAAUCCUAAAAACUCUGCUU	20	513
  CCR5-161	−	GGUGUCGAAAUGAGAAGAAG	20	514
  CCR5-162	−	GAAAUGAGAAGAAGAGGCAC	20	515
  CCR5-163	−	AAAUGAGAAGAAGAGGCACA	20	516
  CCR5-164	−	AGAAGAGGCACAGGGCUGUG	20	517
  CCR5-165	−	UGAUUGUUUAUUUUCUCUUC	20	518
  CCR5-166	−	GAUUGUUUAUUUUCUCUUCU	20	519
  CCR5-167	−	CCUUCUCCUGAACACCUUCC	20	520
  CCR5-168	−	AACACCUUCCAGGAAUUCUU	20	521
  CCR5-169	−	AUAAUUGCAGUAGCUCUAAC	20	522
  CCR5-170	−	UUGCAGUAGCUCUAACAGGU	20	523
  CCR5-171	−	CAGGUUGGACCAAGCUAUGC	20	524
  CCR5-172	−	AUGCAGGUGACAGAGACUCU	20	525
  CCR5-173	−	UGCAGGUGACAGAGACUCUU	20	526
  CCR5-174	−	CCCAUCAUCUAUGCCUUUGU	20	527
  CCR5-175	−	CCAUCAUCUAUGCCUUUGUC	20	528
  CCR5-176	−	CAUCAUCUAUGCCUUUGUCG	20	529
  CCR5-177	−	CUGUUCUAUUUUCCAGCAAG	20	530
  CCR5-178	−	UCAGUUUACACCCGAUCCAC	20	531
  CCR5-179	−	CAGUUUACACCCGAUCCACU	20	532
  CCR5-180	−	AGUUUACACCCGAUCCACUG	20	533
  CCR5-181	−	CACCCGAUCCACUGGGGAGC	20	534
  CCR5-182	−	UGGGGAGCAGGAAAUAUCUG	20	535
  CCR5-183	−	GGGGAGCAGGAAAUAUCUGU	20	536
  CCR5-184	−	AUAUCUGUGGGCUUGUGACA	20	537
  CCR5-185	−	GCUUGUGACACGGACUCAAG	20	538
  CCR5-186	−	CUUGUGACACGGACUCAAGU	20	539
  CCR5-187	−	UGACACGGACUCAAGUGGGC	20	540
  CCR5-188	−	CCCAGUCAGAGUUGUGCACA	20	541
  CCR5-189	−	CUUAGUUUUCAUACACAGCC	20	542
  CCR5-190	−	UUAGUUUUCAUACACAGCCU	20	543
  CCR5-191	−	UUUUCAUACACAGCCUGGGC	20	544
  CCR5-192	−	UUUCAUACACAGCCUGGGCU	20	545
  CCR5-193	−	UUCAUACACAGCCUGGGCUG	20	546
  CCR5-194	−	UCAUACACAGCCUGGGCUGG	20	547
  CCR5-195	−	UACACAGCCUGGGCUGGGGG	20	548
  CCR5-196	−	ACACAGCCUGGGCUGGGGGU	20	549
  CCR5-197	−	CACAGCCUGGGCUGGGGGUG	20	550
  CCR5-198	−	AGCCUGGGCUGGGGGUGGGG	20	551
  CCR5-199	−	GCCUGGGCUGGGGGUGGGGU	20	552
  CCR5-200	−	GGCUGGGGGUGGGGUGGGAG	20	553
  CCR5-201	−	UGGGAGAGGUCUUUUUUAAA	20	554
  CCR5-202	−	AAAGGAAGUUACUGUUAUAG	20	555
  CCR5-203	−	AAGGAAGUUACUGUUAUAGA	20	556
  CCR5-204	−	CUAAGAUUCAUCCAUUUAUU	20	557
  CCR5-205	−	ACAACUUUUUACCUAGUACA	20	558
  CCR5-206	−	CCUAGUACAAGGCAACAUAU	20	559
  CCR5-207	−	GUUGUAAAUGUGUUUAAAAC	20	560
  CCR5-208	−	AACAGGUCUUUGUCUUGCUA	20	561
  CCR5-209	−	ACAGGUCUUUGUCUUGCUAU	20	562
  CCR5-210	−	CAGGUCUUUGUCUUGCUAUG	20	563
  CCR5-211	−	CAUGUGUGAUUUCCCCUCCA	20	564
  CCR5-212	−	GUGAUUUCCCCUCCAAGGUA	20	565
  CCR5-213	−	AGUUUCACUGACUUAGAACC	20	566
  CCR5-214	−	AGAACCAGGCGAGAGACUUG	20	567
  CCR5-215	−	CAGGCGAGAGACUUGUGGCC	20	568
  CCR5-216	−	AGGCGAGAGACUUGUGGCCU	20	569
  CCR5-217	−	GACUUGUGGCCUGGGAGAGC	20	570
  CCR5-218	−	ACUUGUGGCCUGGGAGAGCU	20	571
  CCR5-219	−	CUUGUGGCCUGGGAGAGCUG	20	572
  CCR5-220	−	GGGAAGCUUCUUAAAUGAGA	20	573
  CCR5-221	−	AAAUGAGAAGGAAUUUGAGU	20	574
  CCR5-222	−	UGAGUUGGAUCAUCUAUUGC	20	575
  CCR5-223	−	GCCUCACUGCAAGCACUGCA	20	576
  CCR5-224	−	CCUCACUGCAAGCACUGCAU	20	577
  CCR5-225	−	AAGCACUGCAUGGGCAAGCU	20	578
  CCR5-226	−	UGGGCAAGCUUGGCUGUAGA	20	579
  CCR5-227	−	GCUGUAGAAGGAGACAGAGC	20	580
  CCR5-228	−	UAGAAGGAGACAGAGCUGGU	20	581
  CCR5-229	−	AGAAGGAGACAGAGCUGGUU	20	582
  CCR5-230	−	CAGAGCUGGUUGGGAAGACA	20	583
  CCR5-231	−	AGAGCUGGUUGGGAAGACAU	20	584
  CCR5-232	−	GAGCUGGUUGGGAAGACAUG	20	585
  CCR5-233	−	CUGGUUGGGAAGACAUGGGG	20	586
  CCR5-234	−	UUGGGAAGACAUGGGGAGGA	20	587
  CCR5-235	−	AGACAUGGGGAGGAAGGACA	20	588
  CCR5-236	−	UAGAUCAUGAAGAACCUUGA	20	589
  CCR5-237	−	GUCUAAGUCAUGAGCUGAGC	20	590
  CCR5-238	−	UCUAAGUCAUGAGCUGAGCA	20	591
  CCR5-239	−	UGAGCUGAGCAGGGAGAUCC	20	592
  CCR5-240	−	CUGAGCAGGGAGAUCCUGGU	20	593
  CCR5-241	−	AUCCUGGUUGGUGUUGCAGA	20	594
  CCR5-242	−	GUUGCAGAAGGUUUACUCUG	20	595
  CCR5-243	−	AAGGUUUACUCUGUGGCCAA	20	596
  CCR5-244	−	GUUUACUCUGUGGCCAAAGG	20	597
  CCR5-245	−	UUUACUCUGUGGCCAAAGGA	20	598
  CCR5-246	−	UCUGUGGCCAAAGGAGGGUC	20	599
  CCR5-247	−	UGGCCAAAGGAGGGUCAGGA	20	600
  CCR5-248	−	GUCAGGAAGGAUGAGCAUUU	20	601
  CCR5-249	−	UCAGGAAGGAUGAGCAUUUA	20	602
  CCR5-250	−	AAGGAUGAGCAUUUAGGGCA	20	603
  CCR5-251	−	GGAGACCACCAACAGCCCUC	20	604
  CCR5-252	−	CCACCAACAGCCCUCAGGUC	20	605
  CCR5-253	−	CACCAACAGCCCUCAGGUCA	20	606
  CCR5-254	−	ACAGCCCUCAGGUCAGGGUG	20	607
  CCR5-255	−	CCCUCAGGUCAGGGUGAGGA	20	608
  CCR5-256	−	GAUGGCCUCUGCUAAGCUCA	20	609
  CCR5-257	−	UCUGCUAAGCUCAAGGCGUG	20	610
  CCR5-258	−	CUAAGCUCAAGGCGUGAGGA	20	611
  CCR5-259	−	UAAGCUCAAGGCGUGAGGAU	20	612
  CCR5-260	−	CUCAAGGCGUGAGGAUGGGA	20	613
  CCR5-261	−	AAGGCGUGAGGAUGGGAAGG	20	614
  CCR5-262	−	AGGCGUGAGGAUGGGAAGGA	20	615
  CCR5-263	−	CGUGAGGAUGGGAAGGAGGG	20	616
  CCR5-264	−	GAAGGAGGGAGGUAUUCGUA	20	617
  CCR5-265	−	GAGGGAGGUAUUCGUAAGGA	20	618
  CCR5-266	−	AGGGAGGUAUUCGUAAGGAU	20	619
  CCR5-267	−	AGGUAUUCGUAAGGAUGGGA	20	620
  CCR5-268	−	UAUUCGUAAGGAUGGGAAGG	20	621
  CCR5-269	−	AUUCGUAAGGAUGGGAAGGA	20	622
  CCR5-270	−	CGUAAGGAUGGGAAGGAGGG	20	623
  CCR5-271	−	AGGUAUUCGUGCAGCAUAUG	20	624
  CCR5-272	−	GGAUGCAGAGUCAGCAGAAC	20	625
  CCR5-273	−	GAUGCAGAGUCAGCAGAACU	20	626
  CCR5-274	−	AUGCAGAGUCAGCAGAACUG	20	627
  CCR5-275	−	CAGAGUCAGCAGAACUGGGG	20	628
  CCR5-276	−	CAGCAGAACUGGGGUGGAUU	20	629
  CCR5-277	−	AGCAGAACUGGGGUGGAUUU	20	630
  CCR5-278	−	GAACUGGGGUGGAUUUGGGU	20	631
  CCR5-279	−	GUGGAUUUGGGUUGGAAGUG	20	632
  CCR5-280	−	UGGAUUUGGGUUGGAAGUGA	20	633
  CCR5-281	−	GUUGGAAGUGAGGGUCAGAG	20	634
  CCR5-282	−	UCCCUAGUCUUCAAGCAGAU	20	635
  CCR5-283	−	GAAAAGACAUCAAGCACAGA	20	636
  CCR5-284	−	AAGACAUCAAGCACAGAAGG	20	637
  CCR5-285	−	ACAUCAAGCACAGAAGGAGG	20	638
  CCR5-286	−	UCAAGCACAGAAGGAGGAGG	20	639
  CCR5-287	−	AGCACAGAAGGAGGAGGAGG	20	640
  CCR5-288	−	GAAGGAGGAGGAGGAGGUUU	20	641
  CCR5-289	−	GGUUUAGGUCAAGAAGAAGA	20	642
  CCR5-290	−	AGGUCAAGAAGAAGAUGGAU	20	643
  CCR5-291	−	AGAAGAUGGAUUGGUGUAAA	20	644
  CCR5-292	−	GAUGGAUUGGUGUAAAAGGA	20	645
  CCR5-293	−	AUGGAUUGGUGUAAAAGGAU	20	646
  CCR5-294	−	UUGGUGUAAAAGGAUGGGUC	20	647
  CCR5-295	−	CACAGUCUCACCCAGACUCC	20	648
  CCR5-296	−	CCAUCCCAGCUGAAAUACUG	20	649
  CCR5-297	−	CAUCCCAGCUGAAAUACUGA	20	650
  CCR5-298	−	AUCCCAGCUGAAAUACUGAG	20	651
  CCR5-299	−	UGAAAUACUGAGGGGUCUCC	20	652
  CCR5-300	−	AAUACUGAGGGGUCUCCAGG	20	653
  CCR5-301	−	ACUAGAUUUAUGAAUACACG	20	654
  CCR5-302	−	UUAUGAAUACACGAGGUAUG	20	655
  CCR5-303	−	AUACACGAGGUAUGAGGUCU	20	656
  CCR5-304	−	UCAGCUCACACAUGAGAUCU	20	657
  CCR5-305	−	UCACACAUGAGAUCUAGGUG	20	658
  CCR5-306	−	AUUACCUAGUAGUCAUUUCA	20	659
  CCR5-307	−	UUACCUAGUAGUCAUUUCAU	20	660
  CCR5-308	−	GUAGUCAUUUCAUGGGUUGU	20	661
  CCR5-309	−	UAGUCAUUUCAUGGGUUGUU	20	662
  CCR5-310	−	UCAUUUCAUGGGUUGUUGGG	20	663
  CCR5-311	−	GUUGUUGGGAGGAUUCUAUG	20	664
  CCR5-312	−	GGAUUCUAUGAGGCAACCAC	20	665
  CCR5-313	−	AAACUCUUAGUUACUCAUUC	20	666
  CCR5-314	−	AACUCUUAGUUACUCAUUCA	20	667
  CCR5-315	−	CUGAGCAAAGCAUUGAGCAA	20	668
  CCR5-316	−	UGAGCAAAGCAUUGAGCAAA	20	669
  CCR5-317	−	GAGCAAAGCAUUGAGCAAAG	20	670
  CCR5-318	−	UGAGCAAAGGGGUCCCAUAG	20	671
  CCR5-319	−	AAAGGGGUCCCAUAGAGGUG	20	672
  CCR5-320	−	AAGGGGUCCCAUAGAGGUGA	20	673
  CCR5-321	−	UGCCCAGUGCACACAAGUGU	20	674
  CCR5-322	−	UUCUGCAUUUAACCGUCAAU	20	675
  CCR5-323	−	AUUUAACCGUCAAUAGGCAA	20	676
  CCR5-324	−	UUUAACCGUCAAUAGGCAAA	20	677
  CCR5-325	−	UUAACCGUCAAUAGGCAAAG	20	678
  CCR5-326	−	UAACCGUCAAUAGGCAAAGG	20	679
  CCR5-327	−	AACCGUCAAUAGGCAAAGGG	20	680
  CCR5-328	−	GUCAAUAGGCAAAGGGGGGA	20	681
  CCR5-329	−	UCAAUAGGCAAAGGGGGGAA	20	682
  CCR5-330	−	GGGGAAGGGACAUAUUCAUU	20	683
  CCR5-331	−	CCUCCGUAUUUCAGACUGAA	20	684
  CCR5-332	−	CUCCGUAUUUCAGACUGAAU	20	685
  CCR5-333	−	UCCGUAUUUCAGACUGAAUG	20	686
  CCR5-334	−	CCGUAUUUCAGACUGAAUGG	20	687
  CCR5-335	−	UAUUUCAGACUGAAUGGGGG	20	688
  CCR5-336	−	AUUUCAGACUGAAUGGGGGU	20	689
  CCR5-337	−	UUUCAGACUGAAUGGGGGUG	20	690
  CCR5-338	−	UUCAGACUGAAUGGGGGUGG	20	691
  CCR5-339	−	UCAGACUGAAUGGGGGUGGG	20	692
  CCR5-340	−	CAGACUGAAUGGGGGUGGGG	20	693
  CCR5-341	−	AGACUGAAUGGGGGUGGGGG	20	694
  CCR5-342	−	GGGGGUGGGGGGGGCGCCUU	20	695
  CCR5-343	−	UGAAUAUACCCCUUAGUGUU	20	696
  CCR5-344	−	GAAUAUACCCCUUAGUGUUU	20	697
  CCR5-345	−	UUUGGGUAUAUUCAUUUCAA	20	698
  CCR5-346	−	UUGGGUAUAUUCAUUUCAAA	20	699
  CCR5-347	−	CAUUUCAAAGGGAGAGAGAG	20	700
  CCR5-348	−	ACUUGAGACUGUUUUGAAUU	20	701
  CCR5-349	−	CUUGAGACUGUUUUGAAUUU	20	702
  CCR5-350	−	UUGAGACUGUUUUGAAUUUG	20	703
  CCR5-351	−	UGAGACUGUUUUGAAUUUGG	20	704
  CCR5-352	−	ACUGUUUUGAAUUUGGGGGA	20	705
  CCR5-353	−	GGCUAAAACCAUCAUAGUAC	20	706
  CCR5-354	−	AAACCAUCAUAGUACAGGUA	20	707
  CCR5-355	−	AUCAUAGUACAGGUAAGGUG	20	708
  CCR5-356	−	UCAUAGUACAGGUAAGGUGA	20	709
  CCR5-357	−	UAAGGUGAGGGAAUAGUAAG	20	710
  CCR5-358	−	GUAAGUGGUGAGAACUACUC	20	711
  CCR5-359	−	UAAGUGGUGAGAACUACUCA	20	712
  CCR5-360	−	GAGAACUACUCAGGGAAUGA	20	713
  CCR5-361	−	GAAGGUGUCAGAAUAAUAAG	20	714
  CCR5-362	−	UCUCAGCCUCUGAAUAUGAA	20	715
  CCR5-363	−	AAUAUGAACGGUGAGCAUUG	20	716
  CCR5-364	−	UGAGCAUUGUGGCUGUCAGC	20	717
  CCR5-365	−	CUGUCAGCAGGAAGCAACGA	20	718
  CCR5-366	−	UGUCAGCAGGAAGCAACGAA	20	719
  CCR5-367	−	UUCCUUUUGCUCUUAAGUUG	20	720
  CCR5-368	−	GGAGAGUGCAACAGUAGCAU	20	721
  CCR5-369	−	UAGCAUAGGACCCUACCCUC	20	722
  CCR5-370	−	AGCAUAGGACCCUACCCUCU	20	723
  CCR5-371	−	ACAGUCAGUAUCAAUUC	17	724
  CCR5-372	−	AUUAAAGAUAGUCAUCU	17	725
  CCR5-373	−	UUAAAGAUAGUCAUCUU	17	726
  CCR5-374	−	UAAAGAUAGUCAUCUUG	17	727
  CCR5-375	−	GAUAGUCAUCUUGGGGC	17	728
  CCR5-376	−	CCUGCCGCUGCUUGUCA	17	729
  CCR5-377	−	CAUGGUCAUCUGCUACU	17	730
  CCR5-378	−	AUGGUCAUCUGCUACUC	17	731
  CCR5-379	−	UCCUAAAAACUCUGCUU	17	732
  CCR5-380	−	GUCGAAAUGAGAAGAAG	17	733
  CCR5-381	−	AUGAGAAGAAGAGGCAC	17	734
  CCR5-382	−	UGAGAAGAAGAGGCACA	17	735
  CCR5-383	−	AGAGGCACAGGGCUGUG	17	736
  CCR5-384	−	UUGUUUAUUUUCUCUUC	17	737
  CCR5-385	−	UGUUUAUUUUCUCUUCU	17	738
  CCR5-386	−	UCUCCUGAACACCUUCC	17	739
  CCR5-387	−	ACCUUCCAGGAAUUCUU	17	740
  CCR5-388	−	AUUGCAGUAGCUCUAAC	17	741
  CCR5-389	−	CAGUAGCUCUAACAGGU	17	742
  CCR5-390	−	GUUGGACCAAGCUAUGC	17	743
  CCR5-391	−	CAGGUGACAGAGACUCU	17	744
  CCR5-392	−	AGGUGACAGAGACUCUU	17	745
  CCR5-393	−	AUCAUCUAUGCCUUUGU	17	746
  CCR5-394	−	UCAUCUAUGCCUUUGUC	17	747
  CCR5-395	−	CAUCUAUGCCUUUGUCG	17	748
  CCR5-396	−	UUCUAUUUUCCAGCAAG	17	749
  CCR5-397	−	GUUUACACCCGAUCCAC	17	750
  CCR5-398	−	UUUACACCCGAUCCACU	17	751
  CCR5-399	−	UUACACCCGAUCCACUG	17	752
  CCR5-400	−	CCGAUCCACUGGGGAGC	17	753
  CCR5-401	−	GGAGCAGGAAAUAUCUG	17	754
  CCR5-402	−	GAGCAGGAAAUAUCUGU	17	755
  CCR5-403	−	UCUGUGGGCUUGUGACA	17	756
  CCR5-404	−	UGUGACACGGACUCAAG	17	757
  CCR5-405	−	GUGACACGGACUCAAGU	17	758
  CCR5-406	−	CACGGACUCAAGUGGGC	17	759
  CCR5-407	−	AGUCAGAGUUGUGCACA	17	760
  CCR5-408	−	AGUUUUCAUACACAGCC	17	761
  CCR5-409	−	GUUUUCAUACACAGCCU	17	762
  CCR5-410	−	UCAUACACAGCCUGGGC	17	763
  CCR5-411	−	CAUACACAGCCUGGGCU	17	764
  CCR5-412	−	AUACACAGCCUGGGCUG	17	765
  CCR5-413	−	UACACAGCCUGGGCUGG	17	766
  CCR5-414	−	ACAGCCUGGGCUGGGGG	17	767
  CCR5-415	−	CAGCCUGGGCUGGGGGU	17	768
  CCR5-416	−	AGCCUGGGCUGGGGGUG	17	769
  CCR5-417	−	CUGGGCUGGGGGUGGGG	17	770
  CCR5-418	−	UGGGCUGGGGGUGGGGU	17	771
  CCR5-419	−	UGGGGGUGGGGUGGGAG	17	772
  CCR5-420	−	GAGAGGUCUUUUUUAAA	17	773
  CCR5-421	−	GGAAGUUACUGUUAUAG	17	774
  CCR5-422	−	GAAGUUACUGUUAUAGA	17	775
  CCR5-423	−	AGAUUCAUCCAUUUAUU	17	776
  CCR5-424	−	ACUUUUUACCUAGUACA	17	777
  CCR5-425	−	AGUACAAGGCAACAUAU	17	778
  CCR5-426	−	GUAAAUGUGUUUAAAAC	17	779
  CCR5-427	−	AGGUCUUUGUCUUGCUA	17	780
  CCR5-428	−	GGUCUUUGUCUUGCUAU	17	781
  CCR5-429	−	GUCUUUGUCUUGCUAUG	17	782
  CCR5-430	−	GUGUGAUUUCCCCUCCA	17	783
  CCR5-431	−	AUUUCCCCUCCAAGGUA	17	784
  CCR5-432	−	UUCACUGACUUAGAACC	17	785
  CCR5-433	−	ACCAGGCGAGAGACUUG	17	786
  CCR5-434	−	GCGAGAGACUUGUGGCC	17	787
  CCR5-435	−	CGAGAGACUUGUGGCCU	17	788
  CCR5-436	−	UUGUGGCCUGGGAGAGC	17	789
  CCR5-437	−	UGUGGCCUGGGAGAGCU	17	790
  CCR5-438	−	GUGGCCUGGGAGAGCUG	17	791
  CCR5-439	−	AAGCUUCUUAAAUGAGA	17	792
  CCR5-440	−	UGAGAAGGAAUUUGAGU	17	793
  CCR5-441	−	GUUGGAUCAUCUAUUGC	17	794
  CCR5-442	−	UCACUGCAAGCACUGCA	17	795
  CCR5-443	−	CACUGCAAGCACUGCAU	17	796
  CCR5-444	−	CACUGCAUGGGCAAGCU	17	797
  CCR5-445	−	GCAAGCUUGGCUGUAGA	17	798
  CCR5-446	−	GUAGAAGGAGACAGAGC	17	799
  CCR5-447	−	AAGGAGACAGAGCUGGU	17	800
  CCR5-448	−	AGGAGACAGAGCUGGUU	17	801
  CCR5-449	−	AGCUGGUUGGGAAGACA	17	802
  CCR5-450	−	GCUGGUUGGGAAGACAU	17	803
  CCR5-451	−	CUGGUUGGGAAGACAUG	17	804
  CCR5-452	−	GUUGGGAAGACAUGGGG	17	805
  CCR5-453	−	GGAAGACAUGGGGAGGA	17	806
  CCR5-454	−	CAUGGGGAGGAAGGACA	17	807
  CCR5-455	−	AUCAUGAAGAACCUUGA	17	808
  CCR5-456	−	UAAGUCAUGAGCUGAGC	17	809
  CCR5-457	−	AAGUCAUGAGCUGAGCA	17	810
  CCR5-458	−	GCUGAGCAGGGAGAUCC	17	811
  CCR5-459	−	AGCAGGGAGAUCCUGGU	17	812
  CCR5-460	−	CUGGUUGGUGUUGCAGA	17	813
  CCR5-461	−	GCAGAAGGUUUACUCUG	17	814
  CCR5-462	−	GUUUACUCUGUGGCCAA	17	815
  CCR5-463	−	UACUCUGUGGCCAAAGG	17	816
  CCR5-464	−	ACUCUGUGGCCAAAGGA	17	817
  CCR5-465	−	GUGGCCAAAGGAGGGUC	17	818
  CCR5-466	−	CCAAAGGAGGGUCAGGA	17	819
  CCR5-467	−	AGGAAGGAUGAGCAUUU	17	820
  CCR5-468	−	GGAAGGAUGAGCAUUUA	17	821
  CCR5-469	−	GAUGAGCAUUUAGGGCA	17	822
  CCR5-470	−	GACCACCAACAGCCCUC	17	823
  CCR5-471	−	CCAACAGCCCUCAGGUC	17	824
  CCR5-472	−	CAACAGCCCUCAGGUCA	17	825
  CCR5-473	−	GCCCUCAGGUCAGGGUG	17	826
  CCR5-474	−	UCAGGUCAGGGUGAGGA	17	827
  CCR5-475	−	GGCCUCUGCUAAGCUCA	17	828
  CCR5-476	−	GCUAAGCUCAAGGCGUG	17	829
  CCR5-477	−	AGCUCAAGGCGUGAGGA	17	830
  CCR5-478	−	GCUCAAGGCGUGAGGAU	17	831
  CCR5-479	−	AAGGCGUGAGGAUGGGA	17	832
  CCR5-480	−	GCGUGAGGAUGGGAAGG	17	833
  CCR5-481	−	CGUGAGGAUGGGAAGGA	17	834
  CCR5-482	−	GAGGAUGGGAAGGAGGG	17	835
  CCR5-483	−	GGAGGGAGGUAUUCGUA	17	836
  CCR5-484	−	GGAGGUAUUCGUAAGGA	17	837
  CCR5-485	−	GAGGUAUUCGUAAGGAU	17	838
  CCR5-486	−	UAUUCGUAAGGAUGGGA	17	839
  CCR5-487	−	UCGUAAGGAUGGGAAGG	17	840
  CCR5-488	−	CGUAAGGAUGGGAAGGA	17	841
  CCR5-489	−	AAGGAUGGGAAGGAGGG	17	842
  CCR5-490	−	UAUUCGUGCAGCAUAUG	17	843
  CCR5-491	−	UGCAGAGUCAGCAGAAC	17	844
  CCR5-492	−	GCAGAGUCAGCAGAACU	17	845
  CCR5-493	−	CAGAGUCAGCAGAACUG	17	846
  CCR5-494	−	AGUCAGCAGAACUGGGG	17	847
  CCR5-495	−	CAGAACUGGGGUGGAUU	17	848
  CCR5-496	−	AGAACUGGGGUGGAUUU	17	849
  CCR5-497	−	CUGGGGUGGAUUUGGGU	17	850
  CCR5-498	−	GAUUUGGGUUGGAAGUG	17	851
  CCR5-499	−	AUUUGGGUUGGAAGUGA	17	852
  CCR5-500	−	GGAAGUGAGGGUCAGAG	17	853
  CCR5-501	−	CUAGUCUUCAAGCAGAU	17	854
  CCR5-502	−	AAGACAUCAAGCACAGA	17	855
  CCR5-503	−	ACAUCAAGCACAGAAGG	17	856
  CCR5-504	−	UCAAGCACAGAAGGAGG	17	857
  CCR5-505	−	AGCACAGAAGGAGGAGG	17	858
  CCR5-506	−	ACAGAAGGAGGAGGAGG	17	859
  CCR5-507	−	GGAGGAGGAGGAGGUUU	17	860
  CCR5-508	−	UUAGGUCAAGAAGAAGA	17	861
  CCR5-509	−	UCAAGAAGAAGAUGGAU	17	862
  CCR5-510	−	AGAUGGAUUGGUGUAAA	17	863
  CCR5-511	−	GGAUUGGUGUAAAAGGA	17	864
  CCR5-512	−	GAUUGGUGUAAAAGGAU	17	865
  CCR5-513	−	GUGUAAAAGGAUGGGUC	17	866
  CCR5-514	−	AGUCUCACCCAGACUCC	17	867
  CCR5-515	−	UCCCAGCUGAAAUACUG	17	868
  CCR5-516	−	CCCAGCUGAAAUACUGA	17	869
  CCR5-517	−	CCAGCUGAAAUACUGAG	17	870
  CCR5-518	−	AAUACUGAGGGGUCUCC	17	871
  CCR5-519	−	ACUGAGGGGUCUCCAGG	17	872
  CCR5-520	−	AGAUUUAUGAAUACACG	17	873
  CCR5-521	−	UGAAUACACGAGGUAUG	17	874
  CCR5-522	−	CACGAGGUAUGAGGUCU	17	875
  CCR5-523	−	GCUCACACAUGAGAUCU	17	876
  CCR5-524	−	CACAUGAGAUCUAGGUG	17	877
  CCR5-525	−	ACCUAGUAGUCAUUUCA	17	878
  CCR5-526	−	CCUAGUAGUCAUUUCAU	17	879
  CCR5-527	−	GUCAUUUCAUGGGUUGU	17	880
  CCR5-528	−	UCAUUUCAUGGGUUGUU	17	881
  CCR5-529	−	UUUCAUGGGUUGUUGGG	17	882
  CCR5-530	−	GUUGGGAGGAUUCUAUG	17	883
  CCR5-531	−	UUCUAUGAGGCAACCAC	17	884
  CCR5-532	−	CUCUUAGUUACUCAUUC	17	885
  CCR5-533	−	UCUUAGUUACUCAUUCA	17	886
  CCR5-534	−	AGCAAAGCAUUGAGCAA	17	887
  CCR5-535	−	GCAAAGCAUUGAGCAAA	17	888
  CCR5-536	−	CAAAGCAUUGAGCAAAG	17	889
  CCR5-537	−	GCAAAGGGGUCCCAUAG	17	890
  CCR5-538	−	GGGGUCCCAUAGAGGUG	17	891
  CCR5-539	−	GGGUCCCAUAGAGGUGA	17	892
  CCR5-540	−	CCAGUGCACACAAGUGU	17	893
  CCR5-541	−	UGCAUUUAACCGUCAAU	17	894
  CCR5-542	−	UAACCGUCAAUAGGCAA	17	895
  CCR5-543	−	AACCGUCAAUAGGCAAA	17	896
  CCR5-544	−	ACCGUCAAUAGGCAAAG	17	897
  CCR5-545	−	CCGUCAAUAGGCAAAGG	17	898
  CCR5-546	−	CGUCAAUAGGCAAAGGG	17	899
  CCR5-547	−	AAUAGGCAAAGGGGGGA	17	900
  CCR5-548	−	AUAGGCAAAGGGGGGAA	17	901
  CCR5-549	−	GAAGGGACAUAUUCAUU	17	902
  CCR5-550	−	CCGUAUUUCAGACUGAA	17	903
  CCR5-551	−	CGUAUUUCAGACUGAAU	17	904
  CCR5-552	−	GUAUUUCAGACUGAAUG	17	905
  CCR5-553	−	UAUUUCAGACUGAAUGG	17	906
  CCR5-554	−	UUCAGACUGAAUGGGGG	17	907
  CCR5-555	−	UCAGACUGAAUGGGGGU	17	908
  CCR5-556	−	CAGACUGAAUGGGGGUG	17	909
  CCR5-557	−	AGACUGAAUGGGGGUGG	17	910
  CCR5-558	−	GACUGAAUGGGGGUGGG	17	911
  CCR5-559	−	ACUGAAUGGGGGUGGGG	17	912
  CCR5-560	−	CUGAAUGGGGGUGGGGG	17	913
  CCR5-561	−	GGUGGGGGGGGCGCCUU	17	914
  CCR5-562	−	AUAUACCCCUUAGUGUU	17	915
  CCR5-563	−	UAUACCCCUUAGUGUUU	17	916
  CCR5-564	−	GGGUAUAUUCAUUUCAA	17	917
  CCR5-565	−	GGUAUAUUCAUUUCAAA	17	918
  CCR5-566	−	UUCAAAGGGAGAGAGAG	17	919
  CCR5-567	−	UGAGACUGUUUUGAAUU	17	920
  CCR5-568	−	GAGACUGUUUUGAAUUU	17	921
  CCR5-569	−	AGACUGUUUUGAAUUUG	17	922
  CCR5-570	−	GACUGUUUUGAAUUUGG	17	923
  CCR5-571	−	GUUUUGAAUUUGGGGGA	17	924
  CCR5-572	−	UAAAACCAUCAUAGUAC	17	925
  CCR5-573	−	CCAUCAUAGUACAGGUA	17	926
  CCR5-574	−	AUAGUACAGGUAAGGUG	17	927
  CCR5-575	−	UAGUACAGGUAAGGUGA	17	928
  CCR5-576	−	GGUGAGGGAAUAGUAAG	17	929
  CCR5-577	−	AGUGGUGAGAACUACUC	17	930
  CCR5-578	−	GUGGUGAGAACUACUCA	17	931
  CCR5-579	−	AACUACUCAGGGAAUGA	17	932
  CCR5-580	−	GGUGUCAGAAUAAUAAG	17	933
  CCR5-581	−	CAGCCUCUGAAUAUGAA	17	934
  CCR5-582	−	AUGAACGGUGAGCAUUG	17	935
  CCR5-583	−	GCAUUGUGGCUGUCAGC	17	936
  CCR5-584	−	UCAGCAGGAAGCAACGA	17	937
  CCR5-585	−	CAGCAGGAAGCAACGAA	17	938
  CCR5-586	−	CUUUUGCUCUUAAGUUG	17	939
  CCR5-587	−	GAGUGCAACAGUAGCAU	17	940
  CCR5-588	−	CAUAGGACCCUACCCUC	17	941
  CCR5-589	−	AUAGGACCCUACCCUCU	17	942
  CCR5-590	+	AUGUCAGAAUGUCUUUGACU	20	943
  CCR5-591	+	AUGUCUUUGACUUGGCCCAG	20	944
  CCR5-592	+	UGUCUUUGACUUGGCCCAGA	20	945
  CCR5-593	+	UUUGACUUGGCCCAGAGGGU	20	946
  CCR5-594	+	UUGACUUGGCCCAGAGGGUA	20	947
  CCR5-595	+	CUCCACAACUUAAGAGCAAA	20	948
  CCR5-596	+	UGCUCACCGUUCAUAUUCAG	20	949
  CCR5-597	+	UCACCUUACCUGUACUAUGA	20	950
  CCR5-598	+	AUGAAUAUACCCAAACACUA	20	951
  CCR5-599	+	UGAAUAUACCCAAACACUAA	20	952
  CCR5-600	+	GAAUAUACCCAAACACUAAG	20	953
  CCR5-601	+	AAGGGGUAUAUUCAUUUCAA	20	954
  CCR5-602	+	AGGGGUAUAUUCAUUUCAAA	20	955
  CCR5-603	+	GGUAUAUUCAUUUCAAAGGG	20	956
  CCR5-604	+	GUAUAUUCAUUUCAAAGGGA	20	957
  CCR5-605	+	ACGAUUUUUUCUGUUGCUUC	20	958
  CCR5-606	+	UCUGUUGCUUCUGGUUUGUC	20	959
  CCR5-607	+	GCUUCUGGUUUGUCUGGAGA	20	960
  CCR5-608	+	GUUUGUCUGGAGAAGGCAUC	20	961
  CCR5-609	+	GCAUCUGGAAUAAGUACCUA	20	962
  CCR5-610	+	CCCCCAUUCAGUCUGAAAUA	20	963
  CCR5-611	+	CCAUUCAGUCUGAAAUACGG	20	964
  CCR5-612	+	UCAGUCUGAAAUACGGAGGC	20	965
  CCR5-613	+	GCUGGUAAAUUGUACUUUUG	20	966
  CCR5-614	+	CUGGUAAAUUGUACUUUUGU	20	967
  CCR5-615	+	UUGUACUUUUGUGGGUUUUA	20	968
  CCR5-616	+	UUUGUGGGUUUUAAGGCUCA	20	969
  CCR5-617	+	UUCCCCCCUUUGCCUAUUGA	20	970
  CCR5-618	+	AUACCUACACUUGUGUGCAC	20	971
  CCR5-619	+	UACCUACACUUGUGUGCACU	20	972
  CCR5-620	+	UACACUUGUGUGCACUGGGC	20	973
  CCR5-621	+	AGGCAGCAUCUUAGUUUUUC	20	974
  CCR5-622	+	UCAGGCUUCCCUCACCUCUA	20	975
  CCR5-623	+	CAGGCUUCCCUCACCUCUAU	20	976
  CCR5-624	+	UAUGUGCUAAAUGCUGCCUG	20	977
  CCR5-625	+	CAACCCAUGAAAUGACUACU	20	978
  CCR5-626	+	UCAUAAAUCUAGUCUCCUCC	20	979
  CCR5-627	+	AGACCCCUCAGUAUUUCAGC	20	980
  CCR5-628	+	GACCCCUCAGUAUUUCAGCU	20	981
  CCR5-629	+	CCUCAGUAUUUCAGCUGGGA	20	982
  CCR5-630	+	CUCAGUAUUUCAGCUGGGAU	20	983
  CCR5-631	+	GUAUUUCAGCUGGGAUGGGA	20	984
  CCR5-632	+	GCAUUCAGUGAAAGACAGCC	20	985
  CCR5-633	+	GUGAAAGACAGCCUGGAGUC	20	986
  CCR5-634	+	UGAAAGACAGCCUGGAGUCU	20	987
  CCR5-635	+	CUGUGCUUGAUGUCUUUUCA	20	988
  CCR5-636	+	UGUGCUUGAUGUCUUUUCAA	20	989
  CCR5-637	+	CUCCAAUCUGCUUGAAGACU	20	990
  CCR5-638	+	UCCAAUCUGCUUGAAGACUA	20	991
  CCR5-639	+	UCACGCCUUGAGCUUAGCAG	20	992
  CCR5-640	+	GCCAUCCUCACCCUGACCUG	20	993
  CCR5-641	+	CCAUCCUCACCCUGACCUGA	20	994
  CCR5-642	+	CACCCUGACCUGAGGGCUGU	20	995
  CCR5-643	+	CCUGACCUGAGGGCUGUUGG	20	996
  CCR5-644	+	CAUCCUUCCUGACCCUCCUU	20	997
  CCR5-645	+	AACCUUCUGCAACACCAACC	20	998
  CCR5-646	+	UGCUCAGCUCAUGACUUAGA	20	999
  CCR5-647	+	UAGACGGAGCAAUGCCGUCA	20	1000
  CCR5-648	+	CCCAUGCAGUGCUUGCAGUG	20	1001
  CCR5-649	+	GAAGCUUCCCCAGCUCUCCC	20	1002
  CCR5-650	+	CAGGCCACAAGUCUCUCGCC	20	1003
  CCR5-651	+	GAAACUUAUUAACCAUACCU	20	1004
  CCR5-652	+	ACUUAUUAACCAUACCUUGG	20	1005
  CCR5-653	+	CUUAUUAACCAUACCUUGGA	20	1006
  CCR5-654	+	UUAUUAACCAUACCUUGGAG	20	1007
  CCR5-655	+	CCUAUAUGUUGCCUUGUACU	20	1008
  CCR5-656	+	GUACAUUUCUGAAAUAAUUU	20	1009
  CCR5-657	+	CAAGAAUCAGCAAUUCUCUG	20	1010
  CCR5-658	+	CUUUCUUUUAAAUAUACAUA	20	1011
  CCR5-659	+	AAAUAUACAUAAGGAACUUU	20	1012
  CCR5-660	+	AUAAGGAACUUUCGGAGUGA	20	1013
  CCR5-661	+	UAAGGAACUUUCGGAGUGAA	20	1014
  CCR5-662	+	CAAUAACUUGAUGCAUGUGA	20	1015
  CCR5-663	+	AAUAACUUGAUGCAUGUGAA	20	1016
  CCR5-664	+	AUAACUUGAUGCAUGUGAAG	20	1017
  CCR5-665	+	CAUGUGAAGGGGAGAUAAAA	20	1018
  CCR5-666	+	UUCAUCAACAUAUUUUGAUU	20	1019
  CCR5-667	+	AUUUGGCUUUCUAUAAUUGA	20	1020
  CCR5-668	+	UUUGGCUUUCUAUAAUUGAU	20	1021
  CCR5-669	+	UUAAACAGAUGCCAAAUAAA	20	1022
  CCR5-670	+	UCCCACCCCACCCCCAGCCC	20	1023
  CCR5-671	+	GCCAUGUGCACAACUCUGAC	20	1024
  CCR5-672	+	CCAUGUGCACAACUCUGACU	20	1025
  CCR5-673	+	AGAUAUUUCCUGCUCCCCAG	20	1026
  CCR5-674	+	UUUCCUGCUCCCCAGUGGAU	20	1027
  CCR5-675	+	UUCCUGCUCCCCAGUGGAUC	20	1028
  CCR5-676	+	GUAAACUGAGCUUGCUCGCU	20	1029
  CCR5-677	+	UAAACUGAGCUUGCUCGCUC	20	1030
  CCR5-678	+	CUCGCUCGGGAGCCUCUUGC	20	1031
  CCR5-679	+	ACAGCAUUUGCAGAAGCGUU	20	1032
  CCR5-680	+	AGCGUUUGGCAAUGUGCUUU	20	1033
  CCR5-681	+	GCUUUUGGAAGAAGACUAAG	20	1034
  CCR5-682	+	UCUGAACUUCUCCCCGACAA	20	1035
  CCR5-683	+	CCCGACAAAGGCAUAGAUGA	20	1036
  CCR5-684	+	CCGACAAAGGCAUAGAUGAU	20	1037
  CCR5-685	+	CGACAAAGGCAUAGAUGAUG	20	1038
  CCR5-686	+	UCUCUGUCACCUGCAUAGCU	20	1039
  CCR5-687	+	UAGAGCUACUGCAAUUAUUC	20	1040
  CCR5-688	+	UAUUCAGGCCAAAGAAUUCC	20	1041
  CCR5-689	+	CAGGCCAAAGAAUUCCUGGA	20	1042
  CCR5-690	+	AGAAUUCCUGGAAGGUGUUC	20	1043
  CCR5-691	+	CCUGGAAGGUGUUCAGGAGA	20	1044
  CCR5-692	+	CAGGAGAAGGACAAUGUUGU	20	1045
  CCR5-693	+	AGGAGAAGGACAAUGUUGUA	20	1046
  CCR5-694	+	GAGAAAAUAAACAAUCAUGA	20	1047
  CCR5-695	+	GACACCGAAGCAGAGUUUUU	20	1048
  CCR5-696	+	CAGAUGACCAUGACAAGCAG	20	1049
  CCR5-697	+	UGACCAUGACAAGCAGCGGC	20	1050
  CCR5-698	+	AGAUGACUAUCUUUAAUGUC	20	1051
  CCR5-699	+	CAGAAUUGAUACUGACUGUA	20	1052
  CCR5-700	+	GUAUGGAAAAUGAGAGCUGC	20	1053
  CCR5-701	+	UCAGAAUGUCUUUGACU	17	1054
  CCR5-702	+	UCUUUGACUUGGCCCAG	17	1055
  CCR5-703	+	CUUUGACUUGGCCCAGA	17	1056
  CCR5-704	+	GACUUGGCCCAGAGGGU	17	1057
  CCR5-705	+	ACUUGGCCCAGAGGGUA	17	1058
  CCR5-706	+	CACAACUUAAGAGCAAA	17	1059
  CCR5-707	+	UCACCGUUCAUAUUCAG	17	1060
  CCR5-708	+	CCUUACCUGUACUAUGA	17	1061
  CCR5-709	+	AAUAUACCCAAACACUA	17	1062
  CCR5-710	+	AUAUACCCAAACACUAA	17	1063
  CCR5-711	+	UAUACCCAAACACUAAG	17	1064
  CCR5-712	+	GGGUAUAUUCAUUUCAA	17	1065
  CCR5-713	+	GGUAUAUUCAUUUCAAA	17	1066
  CCR5-714	+	AUAUUCAUUUCAAAGGG	17	1067
  CCR5-715	+	UAUUCAUUUCAAAGGGA	17	1068
  CCR5-716	+	AUUUUUUCUGUUGCUUC	17	1069
  CCR5-717	+	GUUGCUUCUGGUUUGUC	17	1070
  CCR5-718	+	UCUGGUUUGUCUGGAGA	17	1071
  CCR5-719	+	UGUCUGGAGAAGGCAUC	17	1072
  CCR5-720	+	UCUGGAAUAAGUACCUA	17	1073
  CCR5-721	+	CCAUUCAGUCUGAAAUA	17	1074
  CCR5-722	+	UUCAGUCUGAAAUACGG	17	1075
  CCR5-723	+	GUCUGAAAUACGGAGGC	17	1076
  CCR5-724	+	GGUAAAUUGUACUUUUG	17	1077
  CCR5-725	+	GUAAAUUGUACUUUUGU	17	1078
  CCR5-726	+	UACUUUUGUGGGUUUUA	17	1079
  CCR5-727	+	GUGGGUUUUAAGGCUCA	17	1080
  CCR5-728	+	CCCCCUUUGCCUAUUGA	17	1081
  CCR5-729	+	CCUACACUUGUGUGCAC	17	1082
  CCR5-730	+	CUACACUUGUGUGCACU	17	1083
  CCR5-731	+	ACUUGUGUGCACUGGGC	17	1084
  CCR5-732	+	CAGCAUCUUAGUUUUUC	17	1085
  CCR5-733	+	GGCUUCCCUCACCUCUA	17	1086
  CCR5-734	+	GCUUCCCUCACCUCUAU	17	1087
  CCR5-735	+	GUGCUAAAUGCUGCCUG	17	1088
  CCR5-736	+	CCCAUGAAAUGACUACU	17	1089
  CCR5-737	+	UAAAUCUAGUCUCCUCC	17	1090
  CCR5-738	+	CCCCUCAGUAUUUCAGC	17	1091
  CCR5-739	+	CCCUCAGUAUUUCAGCU	17	1092
  CCR5-740	+	CAGUAUUUCAGCUGGGA	17	1093
  CCR5-741	+	AGUAUUUCAGCUGGGAU	17	1094
  CCR5-742	+	UUUCAGCUGGGAUGGGA	17	1095
  CCR5-743	+	UUCAGUGAAAGACAGCC	17	1096
  CCR5-744	+	AAAGACAGCCUGGAGUC	17	1097
  CCR5-745	+	AAGACAGCCUGGAGUCU	17	1098
  CCR5-746	+	UGCUUGAUGUCUUUUCA	17	1099
  CCR5-747	+	GCUUGAUGUCUUUUCAA	17	1100
  CCR5-748	+	CAAUCUGCUUGAAGACU	17	1101
  CCR5-749	+	AAUCUGCUUGAAGACUA	17	1102
  CCR5-750	+	CGCCUUGAGCUUAGCAG	17	1103
  CCR5-751	+	AUCCUCACCCUGACCUG	17	1104
  CCR5-752	+	UCCUCACCCUGACCUGA	17	1105
  CCR5-753	+	CCUGACCUGAGGGCUGU	17	1106
  CCR5-754	+	GACCUGAGGGCUGUUGG	17	1107
  CCR5-755	+	CCUUCCUGACCCUCCUU	17	1108
  CCR5-756	+	CUUCUGCAACACCAACC	17	1109
  CCR5-757	+	UCAGCUCAUGACUUAGA	17	1110
  CCR5-758	+	ACGGAGCAAUGCCGUCA	17	1111
  CCR5-759	+	AUGCAGUGCUUGCAGUG	17	1112
  CCR5-760	+	GCUUCCCCAGCUCUCCC	17	1113
  CCR5-761	+	GCCACAAGUCUCUCGCC	17	1114
  CCR5-762	+	ACUUAUUAACCAUACCU	17	1115
  CCR5-763	+	UAUUAACCAUACCUUGG	17	1116
  CCR5-764	+	AUUAACCAUACCUUGGA	17	1117
  CCR5-765	+	UUAACCAUACCUUGGAG	17	1118
  CCR5-766	+	AUAUGUUGCCUUGUACU	17	1119
  CCR5-767	+	CAUUUCUGAAAUAAUUU	17	1120
  CCR5-768	+	GAAUCAGCAAUUCUCUG	17	1121
  CCR5-769	+	UCUUUUAAAUAUACAUA	17	1122
  CCR5-770	+	UAUACAUAAGGAACUUU	17	1123
  CCR5-771	+	AGGAACUUUCGGAGUGA	17	1124
  CCR5-772	+	GGAACUUUCGGAGUGAA	17	1125
  CCR5-773	+	UAACUUGAUGCAUGUGA	17	1126
  CCR5-774	+	AACUUGAUGCAUGUGAA	17	1127
  CCR5-775	+	ACUUGAUGCAUGUGAAG	17	1128
  CCR5-776	+	GUGAAGGGGAGAUAAAA	17	1129
  CCR5-777	+	AUCAACAUAUUUUGAUU	17	1130
  CCR5-778	+	UGGCUUUCUAUAAUUGA	17	1131
  CCR5-779	+	GGCUUUCUAUAAUUGAU	17	1132
  CCR5-780	+	AACAGAUGCCAAAUAAA	17	1133
  CCR5-781	+	CACCCCACCCCCAGCCC	17	1134
  CCR5-782	+	AUGUGCACAACUCUGAC	17	1135
  CCR5-783	+	UGUGCACAACUCUGACU	17	1136
  CCR5-784	+	UAUUUCCUGCUCCCCAG	17	1137
  CCR5-785	+	CCUGCUCCCCAGUGGAU	17	1138
  CCR5-786	+	CUGCUCCCCAGUGGAUC	17	1139
  CCR5-787	+	AACUGAGCUUGCUCGCU	17	1140
  CCR5-788	+	ACUGAGCUUGCUCGCUC	17	1141
  CCR5-789	+	GCUCGGGAGCCUCUUGC	17	1142
  CCR5-790	+	GCAUUUGCAGAAGCGUU	17	1143
  CCR5-791	+	GUUUGGCAAUGUGCUUU	17	1144
  CCR5-792	+	UUUGGAAGAAGACUAAG	17	1145
  CCR5-793	+	GAACUUCUCCCCGACAA	17	1146
  CCR5-794	+	GACAAAGGCAUAGAUGA	17	1147
  CCR5-795	+	ACAAAGGCAUAGAUGAU	17	1148
  CCR5-796	+	CAAAGGCAUAGAUGAUG	17	1149
  CCR5-797	+	CUGUCACCUGCAUAGCU	17	1150
  CCR5-798	+	AGCUACUGCAAUUAUUC	17	1151
  CCR5-799	+	UCAGGCCAAAGAAUUCC	17	1152
  CCR5-800	+	GCCAAAGAAUUCCUGGA	17	1153
  CCR5-801	+	AUUCCUGGAAGGUGUUC	17	1154
  CCR5-802	+	GGAAGGUGUUCAGGAGA	17	1155
  CCR5-803	+	GAGAAGGACAAUGUUGU	17	1156
  CCR5-804	+	AGAAGGACAAUGUUGUA	17	1157
  CCR5-805	+	AAAAUAAACAAUCAUGA	17	1158
  CCR5-806	+	ACCGAAGCAGAGUUUUU	17	1159
  CCR5-807	+	AUGACCAUGACAAGCAG	17	1160
  CCR5-808	+	CCAUGACAAGCAGCGGC	17	1161
  CCR5-809	+	UGACUAUCUUUAAUGUC	17	1162
  CCR5-810	+	AAUUGAUACUGACUGUA	17	1163
  CCR5-811	+	UGGAAAAUGAGAGCUGC	17	1164

• Table 1E provides targeting domains for knocking out the CCR5 gene. In an embodiment, the targeting domain is the exact complement of the target domain. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that gives double stranded cleavage. Any of the targeting domains in the table can be used with a S. aureus Cas9 single-stranded break nucleases (nickases). In an embodiment, dual targeting is used to create two nicks.

• TABLE 1E
  			Target	SEQ
  	DNA		Site	ID
  gRNA Name	Strand	Targeting Domain	Length	NO

  CCR5-812	−	AUGACAUCAAUUAUUAUACA	20	1165
  CCR5-813	−	UGACAUCAAUUAUUAUACAU	20	1166
  CCR5-814	−	AGCCCUGCCAAAAAAUCAAU	20	1167
  CCR5-815	−	UGGUGUUCAUCUUUGGUUUU	20	1168
  CCR5-816	−	UCCUGAUAAACUGCAAAAGG	20	1169
  CCR5-817	−	UGAUAAACUGCAAAAGGCUG	20	1170
  CCR5-818	−	UUCCUUCUUACUGUCCCCUU	20	1171
  CCR5-819	−	GCUCACUAUGCUGCCGCCCA	20	1172
  CCR5-820	−	CUCACUAUGCUGCCGCCCAG	20	1173
  CCR5-821	−	UGCUGCCGCCCAGUGGGACU	20	1174
  CCR5-822	−	GCUGCCGCCCAGUGGGACUU	20	1175
  CCR5-823	−	UACAAUGUGUCAACUCUUGA	20	1176
  CCR5-824	−	CUAUUUUAUAGGCUUCUUCU	20	1177
  CCR5-825	−	UAUUUUAUAGGCUUCUUCUC	20	1178
  CCR5-826	−	GCUGUGUUUGCUUUAAAAGC	20	1179
  CCR5-827	−	AAAAGCCAGGACGGUCACCU	20	1180
  CCR5-828	−	AAAGCCAGGACGGUCACCUU	20	1181
  CCR5-829	−	GUGGUGACAAGUGUGAUCAC	20	1182
  CCR5-830	−	GGCUGUGUUUGCGUCUCUCC	20	1183
  CCR5-831	−	GCUGUGUUUGCGUCUCUCCC	20	1184
  CCR5-832	−	ACAUCAAUUAUUAUACA	17	1185
  CCR5-833	−	CAUCAAUUAUUAUACAU	17	1186
  CCR5-834	−	CCUGCCAAAAAAUCAAU	17	1187
  CCR5-835	−	UGUUCAUCUUUGGUUUU	17	1188
  CCR5-836	−	UGAUAAACUGCAAAAGG	17	1189
  CCR5-837	−	UAAACUGCAAAAGGCUG	17	1190
  CCR5-838	−	CUUCUUACUGUCCCCUU	17	1191
  CCR5-839	−	CACUAUGCUGCCGCCCA	17	1192
  CCR5-840	−	ACUAUGCUGCCGCCCAG	17	1193
  CCR5-841	−	UGCCGCCCAGUGGGACU	17	1194
  CCR5-842	−	GCCGCCCAGUGGGACUU	17	1195
  CCR5-843	−	AAUGUGUCAACUCUUGA	17	1196
  CCR5-844	−	UUUUAUAGGCUUCUUCU	17	1197
  CCR5-845	−	UUUAUAGGCUUCUUCUC	17	1198
  CCR5-846	−	GUGUUUGCUUUAAAAGC	17	1199
  CCR5-847	−	AGCCAGGACGGUCACCU	17	1200
  CCR5-848	−	GCCAGGACGGUCACCUU	17	1201
  CCR5-849	−	GUGACAAGUGUGAUCAC	17	1202
  CCR5-850	−	UGUGUUUGCGUCUCUCC	17	1203
  CCR5-851	−	GUGUUUGCGUCUCUCCC	17	1204
  CCR5-852	+	GCUUUUAAAGCAAACACAGC	20	1205
  CCR5-853	+	GCCAGGUACCUAUCGAUUGU	20	1206
  CCR5-854	+	CCAGGUACCUAUCGAUUGUC	20	1207
  CCR5-855	+	AGGUACCUAUCGAUUGUCAG	20	1208
  CCR5-856	+	UAUCGAUUGUCAGGAGGAUG	20	1209
  CCR5-857	+	CGAUUGUCAGGAGGAUGAUG	20	1210
  CCR5-858	+	GAGGAUGAUGAAGAAGAUUC	20	1211
  CCR5-859	+	GGAUGAUGAAGAAGAUUCCA	20	1212
  CCR5-860	+	UGAUGAAGAAGAUUCCAGAG	20	1213
  CCR5-861	+	CAGAGAAGAAGCCUAUAAAA	20	1214
  CCR5-862	+	CUAUAAAAUAGAGCCCUGUC	20	1215
  CCR5-863	+	AUUGUAUUUCCAAAGUCCCA	20	1216
  CCR5-864	+	UCCCACUGGGCGGCAGCAUA	20	1217
  CCR5-865	+	GGGCGGCAGCAUAGUGAGCC	20	1218
  CCR5-866	+	CGGCAGCAUAGUGAGCCCAG	20	1219
  CCR5-867	+	GGCAGCAUAGUGAGCCCAGA	20	1220
  CCR5-868	+	GCAGCAUAGUGAGCCCAGAA	20	1221
  CCR5-869	+	UGAGCCCAGAAGGGGACAGU	20	1222
  CCR5-870	+	GCCCAGAAGGGGACAGUAAG	20	1223
  CCR5-871	+	CCCAGAAGGGGACAGUAAGA	20	1224
  CCR5-872	+	AGUAAGAAGGAAAAACAGGU	20	1225
  CCR5-873	+	ACAGGUCAGAGAUGGCCAGG	20	1226
  CCR5-874	+	UUCAGCCUUUUGCAGUUUAU	20	1227
  CCR5-875	+	GCCUUUUGCAGUUUAUCAGG	20	1228
  CCR5-876	+	CUUUUGCAGUUUAUCAGGAU	20	1229
  CCR5-877	+	UGUUGCCCACAAAACCAAAG	20	1230
  CCR5-878	+	AAAACCAAAGAUGAACACCA	20	1231
  CCR5-879	+	CAAAGAUGAACACCAGUGAG	20	1232
  CCR5-880	+	GAUGAACACCAGUGAGUAGA	20	1233
  CCR5-881	+	AUGAACACCAGUGAGUAGAG	20	1234
  CCR5-882	+	ACCAGUGAGUAGAGCGGAGG	20	1235
  CCR5-883	+	CCAGUGAGUAGAGCGGAGGC	20	1236
  CCR5-884	+	GAGUAGAGCGGAGGCAGGAG	20	1237
  CCR5-885	+	GCUUCACAUUGAUUUUUUGG	20	1238
  CCR5-886	+	AUAAUAAUUGAUGUCAUAGA	20	1239
  CCR5-887	+	UUUAAAGCAAACACAGC	17	1240
  CCR5-888	+	AGGUACCUAUCGAUUGU	17	1241
  CCR5-889	+	GGUACCUAUCGAUUGUC	17	1242
  CCR5-890	+	UACCUAUCGAUUGUCAG	17	1243
  CCR5-891	+	CGAUUGUCAGGAGGAUG	17	1244
  CCR5-892	+	UUGUCAGGAGGAUGAUG	17	1245
  CCR5-893	+	GAUGAUGAAGAAGAUUC	17	1246
  CCR5-894	+	UGAUGAAGAAGAUUCCA	17	1247
  CCR5-895	+	UGAAGAAGAUUCCAGAG	17	1248
  CCR5-896	+	AGAAGAAGCCUAUAAAA	17	1249
  CCR5-897	+	UAAAAUAGAGCCCUGUC	17	1250
  CCR5-898	+	GUAUUUCCAAAGUCCCA	17	1251
  CCR5-899	+	CACUGGGCGGCAGCAUA	17	1252
  CCR5-900	+	CGGCAGCAUAGUGAGCC	17	1253
  CCR5-901	+	CAGCAUAGUGAGCCCAG	17	1254
  CCR5-902	+	AGCAUAGUGAGCCCAGA	17	1255
  CCR5-903	+	GCAUAGUGAGCCCAGAA	17	1256
  CCR5-904	+	GCCCAGAAGGGGACAGU	17	1257
  CCR5-905	+	CAGAAGGGGACAGUAAG	17	1258
  CCR5-906	+	AGAAGGGGACAGUAAGA	17	1259
  CCR5-907	+	AAGAAGGAAAAACAGGU	17	1260
  CCR5-908	+	GGUCAGAGAUGGCCAGG	17	1261
  CCR5-909	+	AGCCUUUUGCAGUUUAU	17	1262
  CCR5-910	+	UUUUGCAGUUUAUCAGG	17	1263
  CCR5-911	+	UUGCAGUUUAUCAGGAU	17	1264
  CCR5-912	+	UGCCCACAAAACCAAAG	17	1265
  CCR5-913	+	ACCAAAGAUGAACACCA	17	1266
  CCR5-914	+	AGAUGAACACCAGUGAG	17	1267
  CCR5-915	+	GAACACCAGUGAGUAGA	17	1268
  CCR5-916	+	AACACCAGUGAGUAGAG	17	1269
  CCR5-917	+	AGUGAGUAGAGCGGAGG	17	1270
  CCR5-918	+	GUGAGUAGAGCGGAGGC	17	1271
  CCR5-919	+	UAGAGCGGAGGCAGGAG	17	1272
  CCR5-920	+	UCACAUUGAUUUUUUGG	17	1273
  CCR5-921	+	AUAAUUGAUGUCAUAGA	17	1274
  CCR5-922	−	CCAUACAGUCAGUAUCAAUU	20	1275
  CCR5-923	−	CAUACAGUCAGUAUCAAUUC	20	1276
  CCR5-924	−	ACAGUCAGUAUCAAUUCUGG	20	1277
  CCR5-925	−	AGACAUUAAAGAUAGUCAUC	20	1278
  CCR5-926	−	GACAUUAAAGAUAGUCAUCU	20	1279
  CCR5-927	−	UUGUCAUGGUCAUCUGCUAC	20	1280
  CCR5-928	−	UGUCAUGGUCAUCUGCUACU	20	1281
  CCR5-929	−	GUCAUGGUCAUCUGCUACUC	20	1282
  CCR5-930	−	CUAAAAACUCUGCUUCGGUG	20	1283
  CCR5-931	−	AACUCUGCUUCGGUGUCGAA	20	1284
  CCR5-932	−	CUCUGCUUCGGUGUCGAAAU	20	1285
  CCR5-933	−	UGCUUCGGUGUCGAAAUGAG	20	1286
  CCR5-934	−	UUCGGUGUCGAAAUGAGAAG	20	1287
  CCR5-935	−	CGAAAUGAGAAGAAGAGGCA	20	1288
  CCR5-936	−	AGAAGAAGAGGCACAGGGCU	20	1289
  CCR5-937	−	AUGAUUGUUUAUUUUCUCUU	20	1290
  CCR5-938	−	CCUACAACAUUGUCCUUCUC	20	1291
  CCR5-939	−	UCCUUCUCCUGAACACCUUC	20	1292
  CCR5-940	−	CCUUCUCCUGAACACCUUCC	20	1293
  CCR5-941	−	CCUUCCAGGAAUUCUUUGGC	20	1294
  CCR5-942	−	AUUGCAGUAGCUCUAACAGG	20	1295
  CCR5-943	−	GGACCAAGCUAUGCAGGUGA	20	1296
  CCR5-944	−	UAUGCAGGUGACAGAGACUC	20	1297
  CCR5-945	−	AUGCAGGUGACAGAGACUCU	20	1298
  CCR5-946	−	CCCCAUCAUCUAUGCCUUUG	20	1299
  CCR5-947	−	CCCAUCAUCUAUGCCUUUGU	20	1300
  CCR5-948	−	CCAUCAUCUAUGCCUUUGUC	20	1301
  CCR5-949	−	CAUCAUCUAUGCCUUUGUCG	20	1302
  CCR5-950	−	UCAUCUAUGCCUUUGUCGGG	20	1303
  CCR5-951	−	GCCUUUGUCGGGGAGAAGUU	20	1304
  CCR5-952	−	AUGCUGUUCUAUUUUCCAGC	20	1305
  CCR5-953	−	UAUUUUCCAGCAAGAGGCUC	20	1306
  CCR5-954	−	UUCCAGCAAGAGGCUCCCGA	20	1307
  CCR5-955	−	CUCAGUUUACACCCGAUCCA	20	1308
  CCR5-956	−	UCAGUUUACACCCGAUCCAC	20	1309
  CCR5-957	−	CAGUUUACACCCGAUCCACU	20	1310
  CCR5-958	−	AGUUUACACCCGAUCCACUG	20	1311
  CCR5-959	−	ACACCCGAUCCACUGGGGAG	20	1312
  CCR5-960	−	CACCCGAUCCACUGGGGAGC	20	1313
  CCR5-961	−	CUGGGGAGCAGGAAAUAUCU	20	1314
  CCR5-962	−	AAUAUCUGUGGGCUUGUGAC	20	1315
  CCR5-963	−	GGCUUGUGACACGGACUCAA	20	1316
  CCR5-964	−	AAGUGGGCUGGUGACCCAGU	20	1317
  CCR5-965	−	GCUUAGUUUUCAUACACAGC	20	1318
  CCR5-966	−	GUUUUCAUACACAGCCUGGG	20	1319
  CCR5-967	−	UUUUCAUACACAGCCUGGGC	20	1320
  CCR5-968	−	UUUCAUACACAGCCUGGGCU	20	1321
  CCR5-969	−	AUACACAGCCUGGGCUGGGG	20	1322
  CCR5-970	−	UACACAGCCUGGGCUGGGGG	20	1323
  CCR5-971	−	CAGCCUGGGCUGGGGGUGGG	20	1324
  CCR5-972	−	AGCCUGGGCUGGGGGUGGGG	20	1325
  CCR5-973	−	GCCUGGGCUGGGGGUGGGGU	20	1326
  CCR5-974	−	CUGGGCUGGGGGUGGGGUGG	20	1327
  CCR5-975	−	GUGGGAGAGGUCUUUUUUAA	20	1328
  CCR5-976	−	UGGGAGAGGUCUUUUUUAAA	20	1329
  CCR5-977	−	UUAAAAGGAAGUUACUGUUA	20	1330
  CCR5-978	−	AAAAGGAAGUUACUGUUAUA	20	1331
  CCR5-979	−	UCUUUUAAGCCCAUCAAUUA	20	1332
  CCR5-980	−	AGCCAAAUCAAAAUAUGUUG	20	1333
  CCR5-981	−	UGACAAACUCUCCCUUCACU	20	1334
  CCR5-982	−	AGUUCCUUAUGUAUAUUUAA	20	1335
  CCR5-983	−	GUAUAUUUAAAAGAAAGCCU	20	1336
  CCR5-984	−	AUAUUUAAAAGAAAGCCUCA	20	1337
  CCR5-985	−	CCUCAGAGAAUUGCUGAUUC	20	1338
  CCR5-986	−	UGAUUCUUGAGUUUAGUGAU	20	1339
  CCR5-987	−	CUUGAGUUUAGUGAUCUGAA	20	1340
  CCR5-988	−	CAGAAAUACCAAAAUUAUUU	20	1341
  CCR5-989	−	AAACAGGUCUUUGUCUUGCU	20	1342
  CCR5-990	−	AACAGGUCUUUGUCUUGCUA	20	1343
  CCR5-991	−	ACAGGUCUUUGUCUUGCUAU	20	1344
  CCR5-992	−	CAGGUCUUUGUCUUGCUAUG	20	1345
  CCR5-993	−	GGUCUUUGUCUUGCUAUGGG	20	1346
  CCR5-994	−	UUGCUAUGGGGAGAAAAGAC	20	1347
  CCR5-995	−	AGACAUGAAUAUGAUUAGUA	20	1348
  CCR5-996	−	GUUAAUAAGUUUCACUGACU	20	1349
  CCR5-997	−	UUUCACUGACUUAGAACCAG	20	1350
  CCR5-998	−	UCACUGACUUAGAACCAGGC	20	1351
  CCR5-999	−	CCAGGCGAGAGACUUGUGGC	20	1352
  CCR5-1000	−	CAGGCGAGAGACUUGUGGCC	20	1353
  CCR5-1001	−	AGGCGAGAGACUUGUGGCCU	20	1354
  CCR5-1002	−	GCGAGAGACUUGUGGCCUGG	20	1355
  CCR5-1003	−	AGACUUGUGGCCUGGGAGAG	20	1356
  CCR5-1004	−	GACUUGUGGCCUGGGAGAGC	20	1357
  CCR5-1005	−	ACUUGUGGCCUGGGAGAGCU	20	1358
  CCR5-1006	−	CUUGUGGCCUGGGAGAGCUG	20	1359
  CCR5-1007	−	GAGCUGGGGAAGCUUCUUAA	20	1360
  CCR5-1008	−	GCUGGGGAAGCUUCUUAAAU	20	1361
  CCR5-1009	−	GGGGAAGCUUCUUAAAUGAG	20	1362
  CCR5-1010	−	GGGAAGCUUCUUAAAUGAGA	20	1363
  CCR5-1011	−	CUUCUUAAAUGAGAAGGAAU	20	1364
  CCR5-1012	−	UAAAUGAGAAGGAAUUUGAG	20	1365
  CCR5-1013	−	UCAUCUAUUGCUGGCAAAGA	20	1366
  CCR5-1014	−	AGCCUCACUGCAAGCACUGC	20	1367
  CCR5-1015	−	UGCAUGGGCAAGCUUGGCUG	20	1368
  CCR5-1016	−	AUGGGCAAGCUUGGCUGUAG	20	1369
  CCR5-1017	−	UGGGCAAGCUUGGCUGUAGA	20	1370
  CCR5-1018	−	AGCUUGGCUGUAGAAGGAGA	20	1371
  CCR5-1019	−	GUAGAAGGAGACAGAGCUGG	20	1372
  CCR5-1020	−	UAGAAGGAGACAGAGCUGGU	20	1373
  CCR5-1021	−	AGAAGGAGACAGAGCUGGUU	20	1374
  CCR5-1022	−	ACAGAGCUGGUUGGGAAGAC	20	1375
  CCR5-1023	−	CAGAGCUGGUUGGGAAGACA	20	1376
  CCR5-1024	−	AGAGCUGGUUGGGAAGACAU	20	1377
  CCR5-1025	−	GAGCUGGUUGGGAAGACAUG	20	1378
  CCR5-1026	−	GCUGGUUGGGAAGACAUGGG	20	1379
  CCR5-1027	−	CUGGUUGGGAAGACAUGGGG	20	1380
  CCR5-1028	−	GUUGGGAAGACAUGGGGAGG	20	1381
  CCR5-1029	−	AGGAAGGACAAGGCUAGAUC	20	1382
  CCR5-1030	−	AAGGACAAGGCUAGAUCAUG	20	1383
  CCR5-1031	−	GGCAUUGCUCCGUCUAAGUC	20	1384
  CCR5-1032	−	UGCUCCGUCUAAGUCAUGAG	20	1385
  CCR5-1033	−	CGUCUAAGUCAUGAGCUGAG	20	1386
  CCR5-1034	−	GUCUAAGUCAUGAGCUGAGC	20	1387
  CCR5-1035	−	UCUAAGUCAUGAGCUGAGCA	20	1388
  CCR5-1036	−	GGAGAUCCUGGUUGGUGUUG	20	1389
  CCR5-1037	−	GAAGGUUUACUCUGUGGCCA	20	1390
  CCR5-1038	−	AAGGUUUACUCUGUGGCCAA	20	1391
  CCR5-1039	−	GGUUUACUCUGUGGCCAAAG	20	1392
  CCR5-1040	−	CUCUGUGGCCAAAGGAGGGU	20	1393
  CCR5-1041	−	UCUGUGGCCAAAGGAGGGUC	20	1394
  CCR5-1042	−	GUGGCCAAAGGAGGGUCAGG	20	1395
  CCR5-1043	−	CCAAAGGAGGGUCAGGAAGG	20	1396
  CCR5-1044	−	GGUCAGGAAGGAUGAGCAUU	20	1397
  CCR5-1045	−	GAAGGAUGAGCAUUUAGGGC	20	1398
  CCR5-1046	−	AAGGAUGAGCAUUUAGGGCA	20	1399
  CCR5-1047	−	ACCACCAACAGCCCUCAGGU	20	1400
  CCR5-1048	−	CCAACAGCCCUCAGGUCAGG	20	1401
  CCR5-1049	−	AACAGCCCUCAGGUCAGGGU	20	1402
  CCR5-1050	−	GCCUCUGCUAAGCUCAAGGC	20	1403
  CCR5-1051	−	CUCUGCUAAGCUCAAGGCGU	20	1404
  CCR5-1052	−	GCUAAGCUCAAGGCGUGAGG	20	1405
  CCR5-1053	−	CUAAGCUCAAGGCGUGAGGA	20	1406
  CCR5-1054	−	UAAGCUCAAGGCGUGAGGAU	20	1407
  CCR5-1055	−	GCUCAAGGCGUGAGGAUGGG	20	1408
  CCR5-1056	−	CUCAAGGCGUGAGGAUGGGA	20	1409
  CCR5-1057	−	CAAGGCGUGAGGAUGGGAAG	20	1410
  CCR5-1058	−	AAGGCGUGAGGAUGGGAAGG	20	1411
  CCR5-1059	−	AGGCGUGAGGAUGGGAAGGA	20	1412
  CCR5-1060	−	GGAAGGAGGGAGGUAUUCGU	20	1413
  CCR5-1061	−	GGAGGGAGGUAUUCGUAAGG	20	1414
  CCR5-1062	−	GAGGGAGGUAUUCGUAAGGA	20	1415
  CCR5-1063	−	AGGGAGGUAUUCGUAAGGAU	20	1416
  CCR5-1064	−	GAGGUAUUCGUAAGGAUGGG	20	1417
  CCR5-1065	−	AGGUAUUCGUAAGGAUGGGA	20	1418
  CCR5-1066	−	GUAUUCGUAAGGAUGGGAAG	20	1419
  CCR5-1067	−	UAUUCGUAAGGAUGGGAAGG	20	1420
  CCR5-1068	−	AUUCGUAAGGAUGGGAAGGA	20	1421
  CCR5-1069	−	GGGAGGUAUUCGUGCAGCAU	20	1422
  CCR5-1070	−	GAGGUAUUCGUGCAGCAUAU	20	1423
  CCR5-1071	−	UCGUGCAGCAUAUGAGGAUG	20	1424
  CCR5-1072	−	AUAUGAGGAUGCAGAGUCAG	20	1425
  CCR5-1073	−	AGGAUGCAGAGUCAGCAGAA	20	1426
  CCR5-1074	−	GGAUGCAGAGUCAGCAGAAC	20	1427
  CCR5-1075	−	GCAGAGUCAGCAGAACUGGG	20	1428
  CCR5-1076	−	UCAGCAGAACUGGGGUGGAU	20	1429
  CCR5-1077	−	AGAACUGGGGUGGAUUUGGG	20	1430
  CCR5-1078	−	GAACUGGGGUGGAUUUGGGU	20	1431
  CCR5-1079	−	GGGGUGGAUUUGGGUUGGAA	20	1432
  CCR5-1080	−	GGUGGAUUUGGGUUGGAAGU	20	1433
  CCR5-1081	−	UUUGGGUUGGAAGUGAGGGU	20	1434
  CCR5-1082	−	UGGGUUGGAAGUGAGGGUCA	20	1435
  CCR5-1083	−	GGUUGGAAGUGAGGGUCAGA	20	1436
  CCR5-1084	−	GUUGGAAGUGAGGGUCAGAG	20	1437
  CCR5-1085	−	AGUGAGGGUCAGAGAGGAGU	20	1438
  CCR5-1086	−	UGAGGGUCAGAGAGGAGUCA	20	1439
  CCR5-1087	−	AGGGUCAGAGAGGAGUCAGA	20	1440
  CCR5-1088	−	AUCCCUAGUCUUCAAGCAGA	20	1441
  CCR5-1089	−	UCCCUAGUCUUCAAGCAGAU	20	1442
  CCR5-1090	−	CCUAGUCUUCAAGCAGAUUG	20	1443
  CCR5-1091	−	CAAGCAGAUUGGAGAAACCC	20	1444
  CCR5-1092	−	CCUUGAAAAGACAUCAAGCA	20	1445
  CCR5-1093	−	UGAAAAGACAUCAAGCACAG	20	1446
  CCR5-1094	−	GAAAAGACAUCAAGCACAGA	20	1447
  CCR5-1095	−	AAAGACAUCAAGCACAGAAG	20	1448
  CCR5-1096	−	AAGACAUCAAGCACAGAAGG	20	1449
  CCR5-1097	−	GACAUCAAGCACAGAAGGAG	20	1450
  CCR5-1098	−	ACAUCAAGCACAGAAGGAGG	20	1451
  CCR5-1099	−	AUCAAGCACAGAAGGAGGAG	20	1452
  CCR5-1100	−	UCAAGCACAGAAGGAGGAGG	20	1453
  CCR5-1101	−	AGGAGGAGGAGGUUUAGGUC	20	1454
  CCR5-1102	−	AGGAGGAGGUUUAGGUCAAG	20	1455
  CCR5-1103	−	AGGUUUAGGUCAAGAAGAAG	20	1456
  CCR5-1104	−	AAGAAGAUGGAUUGGUGUAA	20	1457
  CCR5-1105	−	AGAUGGAUUGGUGUAAAAGG	20	1458
  CCR5-1106	−	AAAAGGAUGGGUCUGGUUUG	20	1459
  CCR5-1107	−	AUGGGUCUGGUUUGCAGAGC	20	1460
  CCR5-1108	−	AGACUCCAGGCUGUCUUUCA	20	1461
  CCR5-1109	−	AGAUUUCCUUCCCAUCCCAG	20	1462
  CCR5-1110	−	UUCCCAUCCCAGCUGAAAUA	20	1463
  CCR5-1111	−	CCCAUCCCAGCUGAAAUACU	20	1464
  CCR5-1112	−	CCAUCCCAGCUGAAAUACUG	20	1465
  CCR5-1113	−	CUGAAAUACUGAGGGGUCUC	20	1466
  CCR5-1114	−	UGAAAUACUGAGGGGUCUCC	20	1467
  CCR5-1115	−	AAAUACUGAGGGGUCUCCAG	20	1468
  CCR5-1116	−	AAUACUGAGGGGUCUCCAGG	20	1469
  CCR5-1117	−	UCCAGGAGGAGACUAGAUUU	20	1470
  CCR5-1118	−	GAGACUAGAUUUAUGAAUAC	20	1471
  CCR5-1119	−	GAUUUAUGAAUACACGAGGU	20	1472
  CCR5-1120	−	AAUACACGAGGUAUGAGGUC	20	1473
  CCR5-1121	−	AUACACGAGGUAUGAGGUCU	20	1474
  CCR5-1122	−	GAACAUACUUCAGCUCACAC	20	1475
  CCR5-1123	−	AGCUCACACAUGAGAUCUAG	20	1476
  CCR5-1124	−	CUCACACAUGAGAUCUAGGU	20	1477
  CCR5-1125	−	GAUUACCUAGUAGUCAUUUC	20	1478
  CCR5-1126	−	AGUAGUCAUUUCAUGGGUUG	20	1479
  CCR5-1127	−	GUAGUCAUUUCAUGGGUUGU	20	1480
  CCR5-1128	−	UAGUCAUUUCAUGGGUUGUU	20	1481
  CCR5-1129	−	GUCAUUUCAUGGGUUGUUGG	20	1482
  CCR5-1130	−	UGGGUUGUUGGGAGGAUUCU	20	1483
  CCR5-1131	−	CAAACUCUUAGUUACUCAUU	20	1484
  CCR5-1132	−	AAACUCUUAGUUACUCAUUC	20	1485
  CCR5-1133	−	UUACUCAUUCAGGGAUAGCA	20	1486
  CCR5-1134	−	GGAUAGCACUGAGCAAAGCA	20	1487
  CCR5-1135	−	ACUGAGCAAAGCAUUGAGCA	20	1488
  CCR5-1136	−	CUGAGCAAAGCAUUGAGCAA	20	1489
  CCR5-1137	−	CAUUGAGCAAAGGGGUCCCA	20	1490
  CCR5-1138	−	AGCAAAGGGGUCCCAUAGAG	20	1491
  CCR5-1139	−	CAAAGGGGUCCCAUAGAGGU	20	1492
  CCR5-1140	−	AAAGGGGUCCCAUAGAGGUG	20	1493
  CCR5-1141	−	AAGGGGUCCCAUAGAGGUGA	20	1494
  CCR5-1142	−	CCCAUAGAGGUGAGGGAAGC	20	1495
  CCR5-1143	−	CAUUUAACCGUCAAUAGGCA	20	1496
  CCR5-1144	−	AUUUAACCGUCAAUAGGCAA	20	1497
  CCR5-1145	−	UUUAACCGUCAAUAGGCAAA	20	1498
  CCR5-1146	−	UUAACCGUCAAUAGGCAAAG	20	1499
  CCR5-1147	−	UAACCGUCAAUAGGCAAAGG	20	1500
  CCR5-1148	−	AACCGUCAAUAGGCAAAGGG	20	1501
  CCR5-1149	−	CGUCAAUAGGCAAAGGGGGG	20	1502
  CCR5-1150	−	GUCAAUAGGCAAAGGGGGGA	20	1503
  CCR5-1151	−	GGGGGAAGGGACAUAUUCAU	20	1504
  CCR5-1152	−	GGGGAAGGGACAUAUUCAUU	20	1505
  CCR5-1153	−	UCAUUUGGAAAUAAGCUGCC	20	1506
  CCR5-1154	−	ACCAGCCUCCGUAUUUCAGA	20	1507
  CCR5-1155	−	GCCUCCGUAUUUCAGACUGA	20	1508
  CCR5-1156	−	CCUCCGUAUUUCAGACUGAA	20	1509
  CCR5-1157	−	CUCCGUAUUUCAGACUGAAU	20	1510
  CCR5-1158	−	GUAUUUCAGACUGAAUGGGG	20	1511
  CCR5-1159	−	UAUUUCAGACUGAAUGGGGG	20	1512
  CCR5-1160	−	AUUUCAGACUGAAUGGGGGU	20	1513
  CCR5-1161	−	UUUCAGACUGAAUGGGGGUG	20	1514
  CCR5-1162	−	UUCAGACUGAAUGGGGGUGG	20	1515
  CCR5-1163	−	UCAGACUGAAUGGGGGUGGG	20	1516
  CCR5-1164	−	GAUGCCUUCUCCAGACAAAC	20	1517
  CCR5-1165	−	UCCAGACAAACCAGAAGCAA	20	1518
  CCR5-1166	−	AAAAUCGUCUCUCCCUCCCU	20	1519
  CCR5-1167	−	CGUCUCUCCCUCCCUUUGAA	20	1520
  CCR5-1168	−	AUGAAUAUACCCCUUAGUGU	20	1521
  CCR5-1169	−	GUUUGGGUAUAUUCAUUUCA	20	1522
  CCR5-1170	−	UUUGGGUAUAUUCAUUUCAA	20	1523
  CCR5-1171	−	UUGGGUAUAUUCAUUUCAAA	20	1524
  CCR5-1172	−	GGGUAUAUUCAUUUCAAAGG	20	1525
  CCR5-1173	−	GUAUAUUCAUUUCAAAGGGA	20	1526
  CCR5-1174	−	AUAUUCAUUUCAAAGGGAGA	20	1527
  CCR5-1175	−	AUUCAUUUCAAAGGGAGAGA	20	1528
  CCR5-1176	−	UCAUAUGAUUGUGCACAUAC	20	1529
  CCR5-1177	−	UGCACAUACUUGAGACUGUU	20	1530
  CCR5-1178	−	UACUUGAGACUGUUUUGAAU	20	1531
  CCR5-1179	−	ACUUGAGACUGUUUUGAAUU	20	1532
  CCR5-1180	−	CUUGAGACUGUUUUGAAUUU	20	1533
  CCR5-1181	−	UUGAGACUGUUUUGAAUUUG	20	1534
  CCR5-1182	−	ACCAUCAUAGUACAGGUAAG	20	1535
  CCR5-1183	−	CAUCAUAGUACAGGUAAGGU	20	1536
  CCR5-1184	−	AUCAUAGUACAGGUAAGGUG	20	1537
  CCR5-1185	−	UCAUAGUACAGGUAAGGUGA	20	1538
  CCR5-1186	−	AGGUGAGGGAAUAGUAAGUG	20	1539
  CCR5-1187	−	GUGAGGGAAUAGUAAGUGGU	20	1540
  CCR5-1188	−	AGUAAGUGGUGAGAACUACU	20	1541
  CCR5-1189	−	GUAAGUGGUGAGAACUACUC	20	1542
  CCR5-1190	−	UAAGUGGUGAGAACUACUCA	20	1543
  CCR5-1191	−	UGGUGAGAACUACUCAGGGA	20	1544
  CCR5-1192	−	UACUCAGGGAAUGAAGGUGU	20	1545
  CCR5-1193	−	AAUGAAGGUGUCAGAAUAAU	20	1546
  CCR5-1194	−	GCUACUGACUUUCUCAGCCU	20	1547
  CCR5-1195	−	GACUUUCUCAGCCUCUGAAU	20	1548
  CCR5-1196	−	UCAGCCUCUGAAUAUGAACG	20	1549
  CCR5-1197	−	GUGAGCAUUGUGGCUGUCAG	20	1550
  CCR5-1198	−	UGAGCAUUGUGGCUGUCAGC	20	1551
  CCR5-1199	−	GUGGCUGUCAGCAGGAAGCA	20	1552
  CCR5-1200	−	GCUGUCAGCAGGAAGCAACG	20	1553
  CCR5-1201	−	CUGUCAGCAGGAAGCAACGA	20	1554
  CCR5-1202	−	UGUCAGCAGGAAGCAACGAA	20	1555
  CCR5-1203	−	UUUCCUUUUGCUCUUAAGUU	20	1556
  CCR5-1204	−	UUCCUUUUGCUCUUAAGUUG	20	1557
  CCR5-1205	−	CCUUUUGCUCUUAAGUUGUG	20	1558
  CCR5-1206	−	UGGAGAGUGCAACAGUAGCA	20	1559
  CCR5-1207	−	GUAGCAUAGGACCCUACCCU	20	1560
  CCR5-1208	−	AUUUGCAUAUUCUUAUGUAU	20	1561
  CCR5-1209	−	AUGUGAAAGUUACAAAUUGC	20	1562
  CCR5-1210	−	GAAAGUUACAAAUUGCUUGA	20	1563
  CCR5-1211	−	UACAGUCAGUAUCAAUU	17	1564
  CCR5-1212	−	ACAGUCAGUAUCAAUUC	17	1565
  CCR5-1213	−	GUCAGUAUCAAUUCUGG	17	1566
  CCR5-1214	−	CAUUAAAGAUAGUCAUC	17	1567
  CCR5-1215	−	AUUAAAGAUAGUCAUCU	17	1568
  CCR5-1216	−	UCAUGGUCAUCUGCUAC	17	1569
  CCR5-1217	−	CAUGGUCAUCUGCUACU	17	1570
  CCR5-1218	−	AUGGUCAUCUGCUACUC	17	1571
  CCR5-1219	−	AAAACUCUGCUUCGGUG	17	1572
  CCR5-1220	−	UCUGCUUCGGUGUCGAA	17	1573
  CCR5-1221	−	UGCUUCGGUGUCGAAAU	17	1574
  CCR5-1222	−	UUCGGUGUCGAAAUGAG	17	1575
  CCR5-1223	−	GGUGUCGAAAUGAGAAG	17	1576
  CCR5-1224	−	AAUGAGAAGAAGAGGCA	17	1577
  CCR5-1225	−	AGAAGAGGCACAGGGCU	17	1578
  CCR5-1226	−	AUUGUUUAUUUUCUCUU	17	1579
  CCR5-1227	−	ACAACAUUGUCCUUCUC	17	1580
  CCR5-1228	−	UUCUCCUGAACACCUUC	17	1581
  CCR5-1229	−	UCUCCUGAACACCUUCC	17	1582
  CCR5-1230	−	UCCAGGAAUUCUUUGGC	17	1583
  CCR5-1231	−	GCAGUAGCUCUAACAGG	17	1584
  CCR5-1232	−	CCAAGCUAUGCAGGUGA	17	1585
  CCR5-1233	−	GCAGGUGACAGAGACUC	17	1586
  CCR5-1234	−	CAGGUGACAGAGACUCU	17	1587
  CCR5-1235	−	CAUCAUCUAUGCCUUUG	17	1588
  CCR5-1236	−	AUCAUCUAUGCCUUUGU	17	1589
  CCR5-1237	−	UCAUCUAUGCCUUUGUC	17	1590
  CCR5-1238	−	CAUCUAUGCCUUUGUCG	17	1591
  CCR5-1239	−	UCUAUGCCUUUGUCGGG	17	1592
  CCR5-1240	−	UUUGUCGGGGAGAAGUU	17	1593
  CCR5-1241	−	CUGUUCUAUUUUCCAGC	17	1594
  CCR5-1242	−	UUUCCAGCAAGAGGCUC	17	1595
  CCR5-1243	−	CAGCAAGAGGCUCCCGA	17	1596
  CCR5-1244	−	AGUUUACACCCGAUCCA	17	1597
  CCR5-1245	−	GUUUACACCCGAUCCAC	17	1598
  CCR5-1246	−	UUUACACCCGAUCCACU	17	1599
  CCR5-1247	−	UUACACCCGAUCCACUG	17	1600
  CCR5-1248	−	CCCGAUCCACUGGGGAG	17	1601
  CCR5-1249	−	CCGAUCCACUGGGGAGC	17	1602
  CCR5-1250	−	GGGAGCAGGAAAUAUCU	17	1603
  CCR5-1251	−	AUCUGUGGGCUUGUGAC	17	1604
  CCR5-1252	−	UUGUGACACGGACUCAA	17	1605
  CCR5-1253	−	UGGGCUGGUGACCCAGU	17	1606
  CCR5-1254	−	UAGUUUUCAUACACAGC	17	1607
  CCR5-1255	−	UUCAUACACAGCCUGGG	17	1608
  CCR5-1256	−	UCAUACACAGCCUGGGC	17	1609
  CCR5-1257	−	CAUACACAGCCUGGGCU	17	1610
  CCR5-1258	−	CACAGCCUGGGCUGGGG	17	1611
  CCR5-1259	−	ACAGCCUGGGCUGGGGG	17	1612
  CCR5-1260	−	CCUGGGCUGGGGGUGGG	17	1613
  CCR5-1261	−	CUGGGCUGGGGGUGGGG	17	1614
  CCR5-1262	−	UGGGCUGGGGGUGGGGU	17	1615
  CCR5-1263	−	GGCUGGGGGUGGGGUGG	17	1616
  CCR5-1264	−	GGAGAGGUCUUUUUUAA	17	1617
  CCR5-1265	−	GAGAGGUCUUUUUUAAA	17	1618
  CCR5-1266	−	AAAGGAAGUUACUGUUA	17	1619
  CCR5-1267	−	AGGAAGUUACUGUUAUA	17	1620
  CCR5-1268	−	UUUAAGCCCAUCAAUUA	17	1621
  CCR5-1269	−	CAAAUCAAAAUAUGUUG	17	1622
  CCR5-1270	−	CAAACUCUCCCUUCACU	17	1623
  CCR5-1271	−	UCCUUAUGUAUAUUUAA	17	1624
  CCR5-1272	−	UAUUUAAAAGAAAGCCU	17	1625
  CCR5-1273	−	UUUAAAAGAAAGCCUCA	17	1626
  CCR5-1274	−	CAGAGAAUUGCUGAUUC	17	1627
  CCR5-1275	−	UUCUUGAGUUUAGUGAU	17	1628
  CCR5-1276	−	GAGUUUAGUGAUCUGAA	17	1629
  CCR5-1277	−	AAAUACCAAAAUUAUUU	17	1630
  CCR5-1278	−	CAGGUCUUUGUCUUGCU	17	1631
  CCR5-1279	−	AGGUCUUUGUCUUGCUA	17	1632
  CCR5-1280	−	GGUCUUUGUCUUGCUAU	17	1633
  CCR5-1281	−	GUCUUUGUCUUGCUAUG	17	1634
  CCR5-1282	−	CUUUGUCUUGCUAUGGG	17	1635
  CCR5-1283	−	CUAUGGGGAGAAAAGAC	17	1636
  CCR5-1284	−	CAUGAAUAUGAUUAGUA	17	1637
  CCR5-1285	−	AAUAAGUUUCACUGACU	17	1638
  CCR5-1286	−	CACUGACUUAGAACCAG	17	1639
  CCR5-1287	−	CUGACUUAGAACCAGGC	17	1640
  CCR5-1288	−	GGCGAGAGACUUGUGGC	17	1641
  CCR5-1289	−	GCGAGAGACUUGUGGCC	17	1642
  CCR5-1290	−	CGAGAGACUUGUGGCCU	17	1643
  CCR5-1291	−	AGAGACUUGUGGCCUGG	17	1644
  CCR5-1292	−	CUUGUGGCCUGGGAGAG	17	1645
  CCR5-1293	−	UUGUGGCCUGGGAGAGC	17	1646
  CCR5-1294	−	UGUGGCCUGGGAGAGCU	17	1647
  CCR5-1295	−	GUGGCCUGGGAGAGCUG	17	1648
  CCR5-1296	−	CUGGGGAAGCUUCUUAA	17	1649
  CCR5-1297	−	GGGGAAGCUUCUUAAAU	17	1650
  CCR5-1298	−	GAAGCUUCUUAAAUGAG	17	1651
  CCR5-1299	−	AAGCUUCUUAAAUGAGA	17	1652
  CCR5-1300	−	CUUAAAUGAGAAGGAAU	17	1653
  CCR5-1301	−	AUGAGAAGGAAUUUGAG	17	1654
  CCR5-1302	−	UCUAUUGCUGGCAAAGA	17	1655
  CCR5-1303	−	CUCACUGCAAGCACUGC	17	1656
  CCR5-1304	−	AUGGGCAAGCUUGGCUG	17	1657
  CCR5-1305	−	GGCAAGCUUGGCUGUAG	17	1658
  CCR5-1306	−	GCAAGCUUGGCUGUAGA	17	1659
  CCR5-1307	−	UUGGCUGUAGAAGGAGA	17	1660
  CCR5-1308	−	GAAGGAGACAGAGCUGG	17	1661
  CCR5-1309	−	AAGGAGACAGAGCUGGU	17	1662
  CCR5-1310	−	AGGAGACAGAGCUGGUU	17	1663
  CCR5-1311	−	GAGCUGGUUGGGAAGAC	17	1664
  CCR5-1312	−	AGCUGGUUGGGAAGACA	17	1665
  CCR5-1313	−	GCUGGUUGGGAAGACAU	17	1666
  CCR5-1314	−	CUGGUUGGGAAGACAUG	17	1667
  CCR5-1315	−	GGUUGGGAAGACAUGGG	17	1668
  CCR5-1316	−	GUUGGGAAGACAUGGGG	17	1669
  CCR5-1317	−	GGGAAGACAUGGGGAGG	17	1670
  CCR5-1318	−	AAGGACAAGGCUAGAUC	17	1671
  CCR5-1319	−	GACAAGGCUAGAUCAUG	17	1672
  CCR5-1320	−	AUUGCUCCGUCUAAGUC	17	1673
  CCR5-1321	−	UCCGUCUAAGUCAUGAG	17	1674
  CCR5-1322	−	CUAAGUCAUGAGCUGAG	17	1675
  CCR5-1323	−	UAAGUCAUGAGCUGAGC	17	1676
  CCR5-1324	−	AAGUCAUGAGCUGAGCA	17	1677
  CCR5-1325	−	GAUCCUGGUUGGUGUUG	17	1678
  CCR5-1326	−	GGUUUACUCUGUGGCCA	17	1679
  CCR5-1327	−	GUUUACUCUGUGGCCAA	17	1680
  CCR5-1328	−	UUACUCUGUGGCCAAAG	17	1681
  CCR5-1329	−	UGUGGCCAAAGGAGGGU	17	1682
  CCR5-1330	−	GUGGCCAAAGGAGGGUC	17	1683
  CCR5-1331	−	GCCAAAGGAGGGUCAGG	17	1684
  CCR5-1332	−	AAGGAGGGUCAGGAAGG	17	1685
  CCR5-1333	−	CAGGAAGGAUGAGCAUU	17	1686
  CCR5-1334	−	GGAUGAGCAUUUAGGGC	17	1687
  CCR5-1335	−	GAUGAGCAUUUAGGGCA	17	1688
  CCR5-1336	−	ACCAACAGCCCUCAGGU	17	1689
  CCR5-1337	−	ACAGCCCUCAGGUCAGG	17	1690
  CCR5-1338	−	AGCCCUCAGGUCAGGGU	17	1691
  CCR5-1339	−	UCUGCUAAGCUCAAGGC	17	1692
  CCR5-1340	−	UGCUAAGCUCAAGGCGU	17	1693
  CCR5-1341	−	AAGCUCAAGGCGUGAGG	17	1694
  CCR5-1342	−	AGCUCAAGGCGUGAGGA	17	1695
  CCR5-1343	−	GCUCAAGGCGUGAGGAU	17	1696
  CCR5-1344	−	CAAGGCGUGAGGAUGGG	17	1697
  CCR5-1345	−	AAGGCGUGAGGAUGGGA	17	1698
  CCR5-1346	−	GGCGUGAGGAUGGGAAG	17	1699
  CCR5-1347	−	GCGUGAGGAUGGGAAGG	17	1700
  CCR5-1348	−	CGUGAGGAUGGGAAGGA	17	1701
  CCR5-1349	−	AGGAGGGAGGUAUUCGU	17	1702
  CCR5-1350	−	GGGAGGUAUUCGUAAGG	17	1703
  CCR5-1351	−	GGAGGUAUUCGUAAGGA	17	1704
  CCR5-1352	−	GAGGUAUUCGUAAGGAU	17	1705
  CCR5-1353	−	GUAUUCGUAAGGAUGGG	17	1706
  CCR5-1354	−	UAUUCGUAAGGAUGGGA	17	1707
  CCR5-1355	−	UUCGUAAGGAUGGGAAG	17	1708
  CCR5-1356	−	UCGUAAGGAUGGGAAGG	17	1709
  CCR5-1357	−	CGUAAGGAUGGGAAGGA	17	1710
  CCR5-1358	−	AGGUAUUCGUGCAGCAU	17	1711
  CCR5-1359	−	GUAUUCGUGCAGCAUAU	17	1712
  CCR5-1360	−	UGCAGCAUAUGAGGAUG	17	1713
  CCR5-1361	−	UGAGGAUGCAGAGUCAG	17	1714
  CCR5-1362	−	AUGCAGAGUCAGCAGAA	17	1715
  CCR5-1363	−	UGCAGAGUCAGCAGAAC	17	1716
  CCR5-1364	−	GAGUCAGCAGAACUGGG	17	1717
  CCR5-1365	−	GCAGAACUGGGGUGGAU	17	1718
  CCR5-1366	−	ACUGGGGUGGAUUUGGG	17	1719
  CCR5-1367	−	CUGGGGUGGAUUUGGGU	17	1720
  CCR5-1368	−	GUGGAUUUGGGUUGGAA	17	1721
  CCR5-1369	−	GGAUUUGGGUUGGAAGU	17	1722
  CCR5-1370	−	GGGUUGGAAGUGAGGGU	17	1723
  CCR5-1371	−	GUUGGAAGUGAGGGUCA	17	1724
  CCR5-1372	−	UGGAAGUGAGGGUCAGA	17	1725
  CCR5-1373	−	GGAAGUGAGGGUCAGAG	17	1726
  CCR5-1374	−	GAGGGUCAGAGAGGAGU	17	1727
  CCR5-1375	−	GGGUCAGAGAGGAGUCA	17	1728
  CCR5-1376	−	GUCAGAGAGGAGUCAGA	17	1729
  CCR5-1377	−	CCUAGUCUUCAAGCAGA	17	1730
  CCR5-1378	−	CUAGUCUUCAAGCAGAU	17	1731
  CCR5-1379	−	AGUCUUCAAGCAGAUUG	17	1732
  CCR5-1380	−	GCAGAUUGGAGAAACCC	17	1733
  CCR5-1381	−	UGAAAAGACAUCAAGCA	17	1734
  CCR5-1382	−	AAAGACAUCAAGCACAG	17	1735
  CCR5-1383	−	AAGACAUCAAGCACAGA	17	1736
  CCR5-1384	−	GACAUCAAGCACAGAAG	17	1737
  CCR5-1385	−	ACAUCAAGCACAGAAGG	17	1738
  CCR5-1386	−	AUCAAGCACAGAAGGAG	17	1739
  CCR5-1387	−	UCAAGCACAGAAGGAGG	17	1740
  CCR5-1388	−	AAGCACAGAAGGAGGAG	17	1741
  CCR5-1389	−	AGCACAGAAGGAGGAGG	17	1742
  CCR5-1390	−	AGGAGGAGGUUUAGGUC	17	1743
  CCR5-1391	−	AGGAGGUUUAGGUCAAG	17	1744
  CCR5-1392	−	UUUAGGUCAAGAAGAAG	17	1745
  CCR5-1393	−	AAGAUGGAUUGGUGUAA	17	1746
  CCR5-1394	−	UGGAUUGGUGUAAAAGG	17	1747
  CCR5-1395	−	AGGAUGGGUCUGGUUUG	17	1748
  CCR5-1396	−	GGUCUGGUUUGCAGAGC	17	1749
  CCR5-1397	−	CUCCAGGCUGUCUUUCA	17	1750
  CCR5-1398	−	UUUCCUUCCCAUCCCAG	17	1751
  CCR5-1399	−	CCAUCCCAGCUGAAAUA	17	1752
  CCR5-1400	−	AUCCCAGCUGAAAUACU	17	1753
  CCR5-1401	−	UCCCAGCUGAAAUACUG	17	1754
  CCR5-1402	−	AAAUACUGAGGGGUCUC	17	1755
  CCR5-1403	−	AAUACUGAGGGGUCUCC	17	1756
  CCR5-1404	−	UACUGAGGGGUCUCCAG	17	1757
  CCR5-1405	−	ACUGAGGGGUCUCCAGG	17	1758
  CCR5-1406	−	AGGAGGAGACUAGAUUU	17	1759
  CCR5-1407	−	ACUAGAUUUAUGAAUAC	17	1760
  CCR5-1408	−	UUAUGAAUACACGAGGU	17	1761
  CCR5-1409	−	ACACGAGGUAUGAGGUC	17	1762
  CCR5-1410	−	CACGAGGUAUGAGGUCU	17	1763
  CCR5-1411	−	CAUACUUCAGCUCACAC	17	1764
  CCR5-1412	−	UCACACAUGAGAUCUAG	17	1765
  CCR5-1413	−	ACACAUGAGAUCUAGGU	17	1766
  CCR5-1414	−	UACCUAGUAGUCAUUUC	17	1767
  CCR5-1415	−	AGUCAUUUCAUGGGUUG	17	1768
  CCR5-1416	−	GUCAUUUCAUGGGUUGU	17	1769
  CCR5-1417	−	UCAUUUCAUGGGUUGUU	17	1770
  CCR5-1418	−	AUUUCAUGGGUUGUUGG	17	1771
  CCR5-1419	−	GUUGUUGGGAGGAUUCU	17	1772
  CCR5-1420	−	ACUCUUAGUUACUCAUU	17	1773
  CCR5-1421	−	CUCUUAGUUACUCAUUC	17	1774
  CCR5-1422	−	CUCAUUCAGGGAUAGCA	17	1775
  CCR5-1423	−	UAGCACUGAGCAAAGCA	17	1776
  CCR5-1424	−	GAGCAAAGCAUUGAGCA	17	1777
  CCR5-1425	−	AGCAAAGCAUUGAGCAA	17	1778
  CCR5-1426	−	UGAGCAAAGGGGUCCCA	17	1779
  CCR5-1427	−	AAAGGGGUCCCAUAGAG	17	1780
  CCR5-1428	−	AGGGGUCCCAUAGAGGU	17	1781
  CCR5-1429	−	GGGGUCCCAUAGAGGUG	17	1782
  CCR5-1430	−	GGGUCCCAUAGAGGUGA	17	1783
  CCR5-1431	−	AUAGAGGUGAGGGAAGC	17	1784
  CCR5-1432	−	UUAACCGUCAAUAGGCA	17	1785
  CCR5-1433	−	UAACCGUCAAUAGGCAA	17	1786
  CCR5-1434	−	AACCGUCAAUAGGCAAA	17	1787
  CCR5-1435	−	ACCGUCAAUAGGCAAAG	17	1788
  CCR5-1436	−	CCGUCAAUAGGCAAAGG	17	1789
  CCR5-1437	−	CGUCAAUAGGCAAAGGG	17	1790
  CCR5-1438	−	CAAUAGGCAAAGGGGGG	17	1791
  CCR5-1439	−	AAUAGGCAAAGGGGGGA	17	1792
  CCR5-1440	−	GGAAGGGACAUAUUCAU	17	1793
  CCR5-1441	−	GAAGGGACAUAUUCAUU	17	1794
  CCR5-1442	−	UUUGGAAAUAAGCUGCC	17	1795
  CCR5-1443	−	AGCCUCCGUAUUUCAGA	17	1796
  CCR5-1444	−	UCCGUAUUUCAGACUGA	17	1797
  CCR5-1445	−	CCGUAUUUCAGACUGAA	17	1798
  CCR5-1446	−	CGUAUUUCAGACUGAAU	17	1799
  CCR5-1447	−	UUUCAGACUGAAUGGGG	17	1800
  CCR5-1448	−	UUCAGACUGAAUGGGGG	17	1801
  CCR5-1449	−	UCAGACUGAAUGGGGGU	17	1802
  CCR5-1450	−	CAGACUGAAUGGGGGUG	17	1803
  CCR5-1451	−	AGACUGAAUGGGGGUGG	17	1804
  CCR5-1452	−	GACUGAAUGGGGGUGGG	17	1805
  CCR5-1453	−	GCCUUCUCCAGACAAAC	17	1806
  CCR5-1454	−	AGACAAACCAGAAGCAA	17	1807
  CCR5-1455	−	AUCGUCUCUCCCUCCCU	17	1808
  CCR5-1456	−	CUCUCCCUCCCUUUGAA	17	1809
  CCR5-1457	−	AAUAUACCCCUUAGUGU	17	1810
  CCR5-1458	−	UGGGUAUAUUCAUUUCA	17	1811
  CCR5-1459	−	GGGUAUAUUCAUUUCAA	17	1812
  CCR5-1460	−	GGUAUAUUCAUUUCAAA	17	1813
  CCR5-1461	−	UAUAUUCAUUUCAAAGG	17	1814
  CCR5-1462	−	UAUUCAUUUCAAAGGGA	17	1815
  CCR5-1463	−	UUCAUUUCAAAGGGAGA	17	1816
  CCR5-1464	−	CAUUUCAAAGGGAGAGA	17	1817
  CCR5-1465	−	UAUGAUUGUGCACAUAC	17	1818
  CCR5-1466	−	ACAUACUUGAGACUGUU	17	1819
  CCR5-1467	−	UUGAGACUGUUUUGAAU	17	1820
  CCR5-1468	−	UGAGACUGUUUUGAAUU	17	1821
  CCR5-1469	−	GAGACUGUUUUGAAUUU	17	1822
  CCR5-1470	−	AGACUGUUUUGAAUUUG	17	1823
  CCR5-1471	−	AUCAUAGUACAGGUAAG	17	1824
  CCR5-1472	−	CAUAGUACAGGUAAGGU	17	1825
  CCR5-1473	−	AUAGUACAGGUAAGGUG	17	1826
  CCR5-1474	−	UAGUACAGGUAAGGUGA	17	1827
  CCR5-1475	−	UGAGGGAAUAGUAAGUG	17	1828
  CCR5-1476	−	AGGGAAUAGUAAGUGGU	17	1829
  CCR5-1477	−	AAGUGGUGAGAACUACU	17	1830
  CCR5-1478	−	AGUGGUGAGAACUACUC	17	1831
  CCR5-1479	−	GUGGUGAGAACUACUCA	17	1832
  CCR5-1480	−	UGAGAACUACUCAGGGA	17	1833
  CCR5-1481	−	UCAGGGAAUGAAGGUGU	17	1834
  CCR5-1482	−	GAAGGUGUCAGAAUAAU	17	1835
  CCR5-1483	−	ACUGACUUUCUCAGCCU	17	1836
  CCR5-1484	−	UUUCUCAGCCUCUGAAU	17	1837
  CCR5-1485	−	GCCUCUGAAUAUGAACG	17	1838
  CCR5-1486	−	AGCAUUGUGGCUGUCAG	17	1839
  CCR5-1487	−	GCAUUGUGGCUGUCAGC	17	1840
  CCR5-1488	−	GCUGUCAGCAGGAAGCA	17	1841
  CCR5-1489	−	GUCAGCAGGAAGCAACG	17	1842
  CCR5-1490	−	UCAGCAGGAAGCAACGA	17	1843
  CCR5-1491	−	CAGCAGGAAGCAACGAA	17	1844
  CCR5-1492	−	CCUUUUGCUCUUAAGUU	17	1845
  CCR5-1493	−	CUUUUGCUCUUAAGUUG	17	1846
  CCR5-1494	−	UUUGCUCUUAAGUUGUG	17	1847
  CCR5-1495	−	AGAGUGCAACAGUAGCA	17	1848
  CCR5-1496	−	GCAUAGGACCCUACCCU	17	1849
  CCR5-1497	−	UGCAUAUUCUUAUGUAU	17	1850
  CCR5-1498	−	UGAAAGUUACAAAUUGC	17	1851
  CCR5-1499	−	AGUUACAAAUUGCUUGA	17	1852
  CCR5-1500	+	UUUGUAACUUUCACAUACAU	20	1853
  CCR5-1501	+	AUAUGCAAAUACUAAGAUGU	20	1854
  CCR5-1502	+	AGAAUGUCUUUGACUUGGCC	20	1855
  CCR5-1503	+	AAUGUCUUUGACUUGGCCCA	20	1856
  CCR5-1504	+	CUUUGACUUGGCCCAGAGGG	20	1857
  CCR5-1505	+	UGUUGCACUCUCCACAACUU	20	1858
  CCR5-1506	+	UCUCCACAACUUAAGAGCAA	20	1859
  CCR5-1507	+	CUCCACAACUUAAGAGCAAA	20	1860
  CCR5-1508	+	CAAUGCUCACCGUUCAUAUU	20	1861
  CCR5-1509	+	UCACCGUUCAUAUUCAGAGG	20	1862
  CCR5-1510	+	ACCGUUCAUAUUCAGAGGCU	20	1863
  CCR5-1511	+	UAUUCUGACACCUUCAUUCC	20	1864
  CCR5-1512	+	UCAAGUAUGUGCACAAUCAU	20	1865
  CCR5-1513	+	AUGUGCACAAUCAUAUGAGA	20	1866
  CCR5-1514	+	CACAAUCAUAUGAGACAGAA	20	1867
  CCR5-1515	+	AAAAACCUCUCUCUCUCCCU	20	1868
  CCR5-1516	+	CCUCUCUCUCUCCCUUUGAA	20	1869
  CCR5-1517	+	AAUGAAUAUACCCAAACACU	20	1870
  CCR5-1518	+	AUGAAUAUACCCAAACACUA	20	1871
  CCR5-1519	+	UAAGGGGUAUAUUCAUUUCA	20	1872
  CCR5-1520	+	AAGGGGUAUAUUCAUUUCAA	20	1873
  CCR5-1521	+	AGGGGUAUAUUCAUUUCAAA	20	1874
  CCR5-1522	+	GGGUAUAUUCAUUUCAAAGG	20	1875
  CCR5-1523	+	GGUAUAUUCAUUUCAAAGGG	20	1876
  CCR5-1524	+	GUAUAUUCAUUUCAAAGGGA	20	1877
  CCR5-1525	+	AUAUUCAUUUCAAAGGGAGG	20	1878
  CCR5-1526	+	UUCUGUUGCUUCUGGUUUGU	20	1879
  CCR5-1527	+	UCUGUUGCUUCUGGUUUGUC	20	1880
  CCR5-1528	+	UGUUGCUUCUGGUUUGUCUG	20	1881
  CCR5-1529	+	GGUUUGUCUGGAGAAGGCAU	20	1882
  CCR5-1530	+	GUUUGUCUGGAGAAGGCAUC	20	1883
  CCR5-1531	+	CCCCCCCACCCCCAUUCAGU	20	1884
  CCR5-1532	+	ACCCCCAUUCAGUCUGAAAU	20	1885
  CCR5-1533	+	CCCCCAUUCAGUCUGAAAUA	20	1886
  CCR5-1534	+	GGCUGGUAAAUUGUACUUUU	20	1887
  CCR5-1535	+	UCAAGGCAGCUUAUUUCCAA	20	1888
  CCR5-1536	+	UGCCUAUUGACGGUUAAAUG	20	1889
  CCR5-1537	+	GAUACCUACACUUGUGUGCA	20	1890
  CCR5-1538	+	UUCAGGCUUCCCUCACCUCU	20	1891
  CCR5-1539	+	UCAGGCUUCCCUCACCUCUA	20	1892
  CCR5-1540	+	UGCUUUGCUCAGUGCUAUCC	20	1893
  CCR5-1541	+	UUGCUCAGUGCUAUCCCUGA	20	1894
  CCR5-1542	+	CUAUCCCUGAAUGAGUAACU	20	1895
  CCR5-1543	+	AACUAAGAGUUUGAUGCUUA	20	1896
  CCR5-1544	+	UGCUGCCUGUGGUUGCCUCA	20	1897
  CCR5-1545	+	UAGAAUCCUCCCAACAACCC	20	1898
  CCR5-1546	+	UCCUCACCUAGAUCUCAUGU	20	1899
  CCR5-1547	+	ACCUAGAUCUCAUGUGUGAG	20	1900
  CCR5-1548	+	UUCAUAAAUCUAGUCUCCUC	20	1901
  CCR5-1549	+	UCAUAAAUCUAGUCUCCUCC	20	1902
  CCR5-1550	+	GAGACCCCUCAGUAUUUCAG	20	1903
  CCR5-1551	+	AGACCCCUCAGUAUUUCAGC	20	1904
  CCR5-1552	+	CCCUCAGUAUUUCAGCUGGG	20	1905
  CCR5-1553	+	CCUCAGUAUUUCAGCUGGGA	20	1906
  CCR5-1554	+	CUCAGUAUUUCAGCUGGGAU	20	1907
  CCR5-1555	+	AGUAUUUCAGCUGGGAUGGG	20	1908
  CCR5-1556	+	GUAUUUCAGCUGGGAUGGGA	20	1909
  CCR5-1557	+	CUGGGAUGGGAAGGAAAUCU	20	1910
  CCR5-1558	+	GGGAAGGAAAUCUAUGAAGU	20	1911
  CCR5-1559	+	UAUGAAGUCAGAAGCAUUCA	20	1912
  CCR5-1560	+	AGCAUUCAGUGAAAGACAGC	20	1913
  CCR5-1561	+	GCAUUCAGUGAAAGACAGCC	20	1914
  CCR5-1562	+	AGUGAAAGACAGCCUGGAGU	20	1915
  CCR5-1563	+	AAAGACAGCCUGGAGUCUGG	20	1916
  CCR5-1564	+	UCUGUGCUUGAUGUCUUUUC	20	1917
  CCR5-1565	+	CAAGGGUUUCUCCAAUCUGC	20	1918
  CCR5-1566	+	UCUCCAAUCUGCUUGAAGAC	20	1919
  CCR5-1567	+	CUCCAAUCUGCUUGAAGACU	20	1920
  CCR5-1568	+	UCUGCAUCCUCAUAUGCUGC	20	1921
  CCR5-1569	+	CCUCCCUCCUUCCCAUCCUU	20	1922
  CCR5-1570	+	CUCCUUCCCAUCCUCACGCC	20	1923
  CCR5-1571	+	UCCUCACGCCUUGAGCUUAG	20	1924
  CCR5-1572	+	GAGGCCAUCCUCACCCUGAC	20	1925
  CCR5-1573	+	GGCCAUCCUCACCCUGACCU	20	1926
  CCR5-1574	+	UCCUGACCCUCCUUUGGCCA	20	1927
  CCR5-1575	+	AAACCUUCUGCAACACCAAC	20	1928
  CCR5-1576	+	CUGCUCAGCUCAUGACUUAG	20	1929
  CCR5-1577	+	UGCUCAGCUCAUGACUUAGA	20	1930
  CCR5-1578	+	UUGCCCAUGCAGUGCUUGCA	20	1931
  CCR5-1579	+	ACUCAAAUUCCUUCUCAUUU	20	1932
  CCR5-1580	+	UCUCGCCUGGUUCUAAGUCA	20	1933
  CCR5-1581	+	UGAAACUUAUUAACCAUACC	20	1934
  CCR5-1582	+	GAAACUUAUUAACCAUACCU	20	1935
  CCR5-1583	+	AACUUAUUAACCAUACCUUG	20	1936
  CCR5-1584	+	ACUUAUUAACCAUACCUUGG	20	1937
  CCR5-1585	+	CUUAUUAACCAUACCUUGGA	20	1938
  CCR5-1586	+	UUAUUAACCAUACCUUGGAG	20	1939
  CCR5-1587	+	CCUUGGAGGGGAAAUCACAC	20	1940
  CCR5-1588	+	AGGUAAAAAGUUGUACAUUU	20	1941
  CCR5-1589	+	CUGUUCAGAUCACUAAACUC	20	1942
  CCR5-1590	+	ACUCAAGAAUCAGCAAUUCU	20	1943
  CCR5-1591	+	GCUUUCUUUUAAAUAUACAU	20	1944
  CCR5-1592	+	CUUUCUUUUAAAUAUACAUA	20	1945
  CCR5-1593	+	UAAAUAUACAUAAGGAACUU	20	1946
  CCR5-1594	+	AAAUAUACAUAAGGAACUUU	20	1947
  CCR5-1595	+	AUACAUAAGGAACUUUCGGA	20	1948
  CCR5-1596	+	CAUAAGGAACUUUCGGAGUG	20	1949
  CCR5-1597	+	AUAAGGAACUUUCGGAGUGA	20	1950
  CCR5-1598	+	UAAGGAACUUUCGGAGUGAA	20	1951
  CCR5-1599	+	AGGAACUUUCGGAGUGAAGG	20	1952
  CCR5-1600	+	UUGUCAAUAACUUGAUGCAU	20	1953
  CCR5-1601	+	UCAAUAACUUGAUGCAUGUG	20	1954
  CCR5-1602	+	CAAUAACUUGAUGCAUGUGA	20	1955
  CCR5-1603	+	AAUAACUUGAUGCAUGUGAA	20	1956
  CCR5-1604	+	AUAACUUGAUGCAUGUGAAG	20	1957
  CCR5-1605	+	GAUUUGGCUUUCUAUAAUUG	20	1958
  CCR5-1606	+	UUUAAACAGAUGCCAAAUAA	20	1959
  CCR5-1607	+	AACAGAUGCCAAAUAAAUGG	20	1960
  CCR5-1608	+	ACCCCCAGCCCAGGCUGUGU	20	1961
  CCR5-1609	+	AGCCAUGUGCACAACUCUGA	20	1962
  CCR5-1610	+	UGACUGGGUCACCAGCCCAC	20	1963
  CCR5-1611	+	CAGAUAUUUCCUGCUCCCCA	20	1964
  CCR5-1612	+	AUUUCCUGCUCCCCAGUGGA	20	1965
  CCR5-1613	+	CCCAGUGGAUCGGGUGUAAA	20	1966
  CCR5-1614	+	UGUAAACUGAGCUUGCUCGC	20	1967
  CCR5-1615	+	GUAAACUGAGCUUGCUCGCU	20	1968
  CCR5-1616	+	UAAACUGAGCUUGCUCGCUC	20	1969
  CCR5-1617	+	GCUCGCUCGGGAGCCUCUUG	20	1970
  CCR5-1618	+	CUCGCUCGGGAGCCUCUUGC	20	1971
  CCR5-1619	+	GGGAGCCUCUUGCUGGAAAA	20	1972
  CCR5-1620	+	GGAAAAUAGAACAGCAUUUG	20	1973
  CCR5-1621	+	AAGCGUUUGGCAAUGUGCUU	20	1974
  CCR5-1622	+	AGCGUUUGGCAAUGUGCUUU	20	1975
  CCR5-1623	+	GUUUGGCAAUGUGCUUUUGG	20	1976
  CCR5-1624	+	UGUGCUUUUGGAAGAAGACU	20	1977
  CCR5-1625	+	AGAAGACUAAGAGGUAGUUU	20	1978
  CCR5-1626	+	CCCCGACAAAGGCAUAGAUG	20	1979
  CCR5-1627	+	CCCGACAAAGGCAUAGAUGA	20	1980
  CCR5-1628	+	AUGCAGCAGUGCGUCAUCCC	20	1981
  CCR5-1629	+	CAUAGCUUGGUCCAACCUGU	20	1982
  CCR5-1630	+	UACUGCAAUUAUUCAGGCCA	20	1983
  CCR5-1631	+	UUAUUCAGGCCAAAGAAUUC	20	1984
  CCR5-1632	+	UAUUCAGGCCAAAGAAUUCC	20	1985
  CCR5-1633	+	AAGAAUUCCUGGAAGGUGUU	20	1986
  CCR5-1634	+	AGAAUUCCUGGAAGGUGUUC	20	1987
  CCR5-1635	+	AAUUCCUGGAAGGUGUUCAG	20	1988
  CCR5-1636	+	UCCUGGAAGGUGUUCAGGAG	20	1989
  CCR5-1637	+	UCAGGAGAAGGACAAUGUUG	20	1990
  CCR5-1638	+	CAGGAGAAGGACAAUGUUGU	20	1991
  CCR5-1639	+	AGGAGAAGGACAAUGUUGUA	20	1992
  CCR5-1640	+	GGACAAUGUUGUAGGGAGCC	20	1993
  CCR5-1641	+	CAAUGUUGUAGGGAGCCCAG	20	1994
  CCR5-1642	+	AUGUUGUAGGGAGCCCAGAA	20	1995
  CCR5-1643	+	GAAAAUAAACAAUCAUGAUG	20	1996
  CCR5-1644	+	CUCUUCUUCUCAUUUCGACA	20	1997
  CCR5-1645	+	UUCUCAUUUCGACACCGAAG	20	1998
  CCR5-1646	+	CGACACCGAAGCAGAGUUUU	20	1999
  CCR5-1647	+	AAGCAGAGUUUUUAGGAUUC	20	2000
  CCR5-1648	+	AUGACCAUGACAAGCAGCGG	20	2001
  CCR5-1649	+	AAGAUGACUAUCUUUAAUGU	20	2002
  CCR5-1650	+	AGAUGACUAUCUUUAAUGUC	20	2003
  CCR5-1651	+	UUAAUGUCUGGAAAUUCUUC	20	2004
  CCR5-1652	+	CCAGAAUUGAUACUGACUGU	20	2005
  CCR5-1653	+	CAGAAUUGAUACUGACUGUA	20	2006
  CCR5-1654	+	UGAUACUGACUGUAUGGAAA	20	2007
  CCR5-1655	+	AUACUGACUGUAUGGAAAAU	20	2008
  CCR5-1656	+	AAAUGAGAGCUGCAGGUGUA	20	2009
  CCR5-1657	+	GUGUAAUGAAGACCUUCUUU	20	2010
  CCR5-1658	+	GUAACUUUCACAUACAU	17	2011
  CCR5-1659	+	UGCAAAUACUAAGAUGU	17	2012
  CCR5-1660	+	AUGUCUUUGACUUGGCC	17	2013
  CCR5-1661	+	GUCUUUGACUUGGCCCA	17	2014
  CCR5-1662	+	UGACUUGGCCCAGAGGG	17	2015
  CCR5-1663	+	UGCACUCUCCACAACUU	17	2016
  CCR5-1664	+	CCACAACUUAAGAGCAA	17	2017
  CCR5-1665	+	CACAACUUAAGAGCAAA	17	2018
  CCR5-1666	+	UGCUCACCGUUCAUAUU	17	2019
  CCR5-1667	+	CCGUUCAUAUUCAGAGG	17	2020
  CCR5-1668	+	GUUCAUAUUCAGAGGCU	17	2021
  CCR5-1669	+	UCUGACACCUUCAUUCC	17	2022
  CCR5-1670	+	AGUAUGUGCACAAUCAU	17	2023
  CCR5-1671	+	UGCACAAUCAUAUGAGA	17	2024
  CCR5-1672	+	AAUCAUAUGAGACAGAA	17	2025
  CCR5-1673	+	AACCUCUCUCUCUCCCU	17	2026
  CCR5-1674	+	CUCUCUCUCCCUUUGAA	17	2027
  CCR5-1675	+	GAAUAUACCCAAACACU	17	2028
  CCR5-1676	+	AAUAUACCCAAACACUA	17	2029
  CCR5-1677	+	GGGGUAUAUUCAUUUCA	17	2030
  CCR5-1678	+	GGGUAUAUUCAUUUCAA	17	2031
  CCR5-1679	+	GGUAUAUUCAUUUCAAA	17	2032
  CCR5-1680	+	UAUAUUCAUUUCAAAGG	17	2033
  CCR5-1681	+	AUAUUCAUUUCAAAGGG	17	2034
  CCR5-1682	+	UAUUCAUUUCAAAGGGA	17	2035
  CCR5-1683	+	UUCAUUUCAAAGGGAGG	17	2036
  CCR5-1684	+	UGUUGCUUCUGGUUUGU	17	2037
  CCR5-1685	+	GUUGCUUCUGGUUUGUC	17	2038
  CCR5-1686	+	UGCUUCUGGUUUGUCUG	17	2039
  CCR5-1687	+	UUGUCUGGAGAAGGCAU	17	2040
  CCR5-1688	+	UGUCUGGAGAAGGCAUC	17	2041
  CCR5-1689	+	CCCCACCCCCAUUCAGU	17	2042
  CCR5-1690	+	CCCAUUCAGUCUGAAAU	17	2043
  CCR5-1691	+	CCAUUCAGUCUGAAAUA	17	2044
  CCR5-1692	+	UGGUAAAUUGUACUUUU	17	2045
  CCR5-1693	+	AGGCAGCUUAUUUCCAA	17	2046
  CCR5-1694	+	CUAUUGACGGUUAAAUG	17	2047
  CCR5-1695	+	ACCUACACUUGUGUGCA	17	2048
  CCR5-1696	+	AGGCUUCCCUCACCUCU	17	2049
  CCR5-1697	+	GGCUUCCCUCACCUCUA	17	2050
  CCR5-1698	+	UUUGCUCAGUGCUAUCC	17	2051
  CCR5-1699	+	CUCAGUGCUAUCCCUGA	17	2052
  CCR5-1700	+	UCCCUGAAUGAGUAACU	17	2053
  CCR5-1701	+	UAAGAGUUUGAUGCUUA	17	2054
  CCR5-1702	+	UGCCUGUGGUUGCCUCA	17	2055
  CCR5-1703	+	AAUCCUCCCAACAACCC	17	2056
  CCR5-1704	+	UCACCUAGAUCUCAUGU	17	2057
  CCR5-1705	+	UAGAUCUCAUGUGUGAG	17	2058
  CCR5-1706	+	AUAAAUCUAGUCUCCUC	17	2059
  CCR5-1707	+	UAAAUCUAGUCUCCUCC	17	2060
  CCR5-1708	+	ACCCCUCAGUAUUUCAG	17	2061
  CCR5-1709	+	CCCCUCAGUAUUUCAGC	17	2062
  CCR5-1710	+	UCAGUAUUUCAGCUGGG	17	2063
  CCR5-1711	+	CAGUAUUUCAGCUGGGA	17	2064
  CCR5-1712	+	AGUAUUUCAGCUGGGAU	17	2065
  CCR5-1713	+	AUUUCAGCUGGGAUGGG	17	2066
  CCR5-1714	+	UUUCAGCUGGGAUGGGA	17	2067
  CCR5-1715	+	GGAUGGGAAGGAAAUCU	17	2068
  CCR5-1716	+	AAGGAAAUCUAUGAAGU	17	2069
  CCR5-1717	+	GAAGUCAGAAGCAUUCA	17	2070
  CCR5-1718	+	AUUCAGUGAAAGACAGC	17	2071
  CCR5-1719	+	UUCAGUGAAAGACAGCC	17	2072
  CCR5-1720	+	GAAAGACAGCCUGGAGU	17	2073
  CCR5-1721	+	GACAGCCUGGAGUCUGG	17	2074
  CCR5-1722	+	GUGCUUGAUGUCUUUUC	17	2075
  CCR5-1723	+	GGGUUUCUCCAAUCUGC	17	2076
  CCR5-1724	+	CCAAUCUGCUUGAAGAC	17	2077
  CCR5-1725	+	CAAUCUGCUUGAAGACU	17	2078
  CCR5-1726	+	GCAUCCUCAUAUGCUGC	17	2079
  CCR5-1727	+	CCCUCCUUCCCAUCCUU	17	2080
  CCR5-1728	+	CUUCCCAUCCUCACGCC	17	2081
  CCR5-1729	+	UCACGCCUUGAGCUUAG	17	2082
  CCR5-1730	+	GCCAUCCUCACCCUGAC	17	2083
  CCR5-1731	+	CAUCCUCACCCUGACCU	17	2084
  CCR5-1732	+	UGACCCUCCUUUGGCCA	17	2085
  CCR5-1733	+	CCUUCUGCAACACCAAC	17	2086
  CCR5-1734	+	CUCAGCUCAUGACUUAG	17	2087
  CCR5-1735	+	UCAGCUCAUGACUUAGA	17	2088
  CCR5-1736	+	CCCAUGCAGUGCUUGCA	17	2089
  CCR5-1737	+	CAAAUUCCUUCUCAUUU	17	2090
  CCR5-1738	+	CGCCUGGUUCUAAGUCA	17	2091
  CCR5-1739	+	AACUUAUUAACCAUACC	17	2092
  CCR5-1740	+	ACUUAUUAACCAUACCU	17	2093
  CCR5-1741	+	UUAUUAACCAUACCUUG	17	2094
  CCR5-1742	+	UAUUAACCAUACCUUGG	17	2095
  CCR5-1743	+	AUUAACCAUACCUUGGA	17	2096
  CCR5-1744	+	UUAACCAUACCUUGGAG	17	2097
  CCR5-1745	+	UGGAGGGGAAAUCACAC	17	2098
  CCR5-1746	+	UAAAAAGUUGUACAUUU	17	2099
  CCR5-1747	+	UUCAGAUCACUAAACUC	17	2100
  CCR5-1748	+	CAAGAAUCAGCAAUUCU	17	2101
  CCR5-1749	+	UUCUUUUAAAUAUACAU	17	2102
  CCR5-1750	+	UCUUUUAAAUAUACAUA	17	2103
  CCR5-1751	+	AUAUACAUAAGGAACUU	17	2104
  CCR5-1752	+	UAUACAUAAGGAACUUU	17	2105
  CCR5-1753	+	CAUAAGGAACUUUCGGA	17	2106
  CCR5-1754	+	AAGGAACUUUCGGAGUG	17	2107
  CCR5-1755	+	AGGAACUUUCGGAGUGA	17	2108
  CCR5-1756	+	GGAACUUUCGGAGUGAA	17	2109
  CCR5-1757	+	AACUUUCGGAGUGAAGG	17	2110
  CCR5-1758	+	UCAAUAACUUGAUGCAU	17	2111
  CCR5-1759	+	AUAACUUGAUGCAUGUG	17	2112
  CCR5-1760	+	UAACUUGAUGCAUGUGA	17	2113
  CCR5-1761	+	AACUUGAUGCAUGUGAA	17	2114
  CCR5-1762	+	ACUUGAUGCAUGUGAAG	17	2115
  CCR5-1763	+	UUGGCUUUCUAUAAUUG	17	2116
  CCR5-1764	+	AAACAGAUGCCAAAUAA	17	2117
  CCR5-1765	+	AGAUGCCAAAUAAAUGG	17	2118
  CCR5-1766	+	CCCAGCCCAGGCUGUGU	17	2119
  CCR5-1767	+	CAUGUGCACAACUCUGA	17	2120
  CCR5-1768	+	CUGGGUCACCAGCCCAC	17	2121
  CCR5-1769	+	AUAUUUCCUGCUCCCCA	17	2122
  CCR5-1770	+	UCCUGCUCCCCAGUGGA	17	2123
  CCR5-1771	+	AGUGGAUCGGGUGUAAA	17	2124
  CCR5-1772	+	AAACUGAGCUUGCUCGC	17	2125
  CCR5-1773	+	AACUGAGCUUGCUCGCU	17	2126
  CCR5-1774	+	ACUGAGCUUGCUCGCUC	17	2127
  CCR5-1775	+	CGCUCGGGAGCCUCUUG	17	2128
  CCR5-1776	+	GCUCGGGAGCCUCUUGC	17	2129
  CCR5-1777	+	AGCCUCUUGCUGGAAAA	17	2130
  CCR5-1778	+	AAAUAGAACAGCAUUUG	17	2131
  CCR5-1779	+	CGUUUGGCAAUGUGCUU	17	2132
  CCR5-1780	+	GUUUGGCAAUGUGCUUU	17	2133
  CCR5-1781	+	UGGCAAUGUGCUUUUGG	17	2134
  CCR5-1782	+	GCUUUUGGAAGAAGACU	17	2135
  CCR5-1783	+	AGACUAAGAGGUAGUUU	17	2136
  CCR5-1784	+	CGACAAAGGCAUAGAUG	17	2137
  CCR5-1785	+	GACAAAGGCAUAGAUGA	17	2138
  CCR5-1786	+	CAGCAGUGCGUCAUCCC	17	2139
  CCR5-1787	+	AGCUUGGUCCAACCUGU	17	2140
  CCR5-1788	+	UGCAAUUAUUCAGGCCA	17	2141
  CCR5-1789	+	UUCAGGCCAAAGAAUUC	17	2142
  CCR5-1790	+	UCAGGCCAAAGAAUUCC	17	2143
  CCR5-1791	+	AAUUCCUGGAAGGUGUU	17	2144
  CCR5-1792	+	AUUCCUGGAAGGUGUUC	17	2145
  CCR5-1793	+	UCCUGGAAGGUGUUCAG	17	2146
  CCR5-1794	+	UGGAAGGUGUUCAGGAG	17	2147
  CCR5-1795	+	GGAGAAGGACAAUGUUG	17	2148
  CCR5-1796	+	GAGAAGGACAAUGUUGU	17	2149
  CCR5-1797	+	AGAAGGACAAUGUUGUA	17	2150
  CCR5-1798	+	CAAUGUUGUAGGGAGCC	17	2151
  CCR5-1799	+	UGUUGUAGGGAGCCCAG	17	2152
  CCR5-1800	+	UUGUAGGGAGCCCAGAA	17	2153
  CCR5-1801	+	AAUAAACAAUCAUGAUG	17	2154
  CCR5-1802	+	UUCUUCUCAUUUCGACA	17	2155
  CCR5-1803	+	UCAUUUCGACACCGAAG	17	2156
  CCR5-1804	+	CACCGAAGCAGAGUUUU	17	2157
  CCR5-1805	+	CAGAGUUUUUAGGAUUC	17	2158
  CCR5-1806	+	ACCAUGACAAGCAGCGG	17	2159
  CCR5-1807	+	AUGACUAUCUUUAAUGU	17	2160
  CCR5-1808	+	UGACUAUCUUUAAUGUC	17	2161
  CCR5-1809	+	AUGUCUGGAAAUUCUUC	17	2162
  CCR5-1810	+	GAAUUGAUACUGACUGU	17	2163
  CCR5-1811	+	AAUUGAUACUGACUGUA	17	2164
  CCR5-1812	+	UACUGACUGUAUGGAAA	17	2165
  CCR5-1813	+	CUGACUGUAUGGAAAAU	17	2166
  CCR5-1814	+	UGAGAGCUGCAGGUGUA	17	2167
  CCR5-1815	+	UAAUGAAGACCUUCUUU	17	2168

• Table 1F provides exemplary targeting domains for knocking out the CCR5 gene. In an embodiment, the targeting domain is the exact complement of the target domain. Any of the targeting domains in the table can be used with an N. meningitides Cas9 molecule that gives double stranded cleavage. Any of the targeting domains in the table can be used with an N. meningitides Cas9 single-stranded break nucleases (nickases). In an embodiment, dual targeting is used to create two nicks.

• TABLE 1F
  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-1816	+	AUGGACGACAGCCAGGUACC	20	2169
  CCR5-1817	+	GAUUGUCAGGAGGAUGAUGA	20	2170
  CCR5-1818	+	GAGCGGAGGCAGGAGGCGGG	20	2171
  CCR5-1819	+	GCGGGCUGCGAUUUGCUUCA	20	2172
  CCR5-1820	+	CGAUGUAUAAUAAUUGAUGU	20	2173
  CCR5-1821	+	GACGACAGCCAGGUACC	17	2174
  CCR5-1822	+	UGUCAGGAGGAUGAUGA	17	2175
  CCR5-1823	+	CGGAGGCAGGAGGCGGG	17	2176
  CCR5-1824	+	GGCUGCGAUUUGCUUCA	17	2177
  CCR5-1825	+	UGUAUAAUAAUUGAUGU	17	2178
  CCR5-1826	−	UGUGAGGCUUAUCUUCACCA	20	2179
  CCR5-1827	−	AAGUUACUGUUAUAGAGGGU	20	2180
  CCR5-1828	−	UUUAUUUGGCAUCUGUUUAA	20	2181
  CCR5-1829	−	AAAAGAAAGCCUCAGAGAAU	20	2182
  CCR5-1830	−	UAUGGGGAGAAAAGACAUGA	20	2183
  CCR5-1831	−	AAAGAAAUGACACUUUUCAU	20	2184
  CCR5-1832	−	UGCAGAGUCAGCAGAACUGG	20	2185
  CCR5-1833	−	GAGAGAAUCCCUAGUCUUCA	20	2186
  CCR5-1834	−	GAGGUUUAGGUCAAGAAGAA	20	2187
  CCR5-1835	−	UCACUGAAUGCUUCUGACUU	20	2188
  CCR5-1836	−	UGAGGGGUCUCCAGGAGGAG	20	2189
  CCR5-1837	−	GCUCACACAUGAGAUCUAGG	20	2190
  CCR5-1838	−	ACACAUGAGAUCUAGGUGAG	20	2191
  CCR5-1839	−	AGUCAUUUCAUGGGUUGUUG	20	2192
  CCR5-1840	−	GUUUUUUUCUGUUCUGUCUC	20	2193
  CCR5-1841	−	GAGGCUUAUCUUCACCA	17	2194
  CCR5-1842	−	UUACUGUUAUAGAGGGU	17	2195
  CCR5-1843	−	AUUUGGCAUCUGUUUAA	17	2196
  CCR5-1844	−	AGAAAGCCUCAGAGAAU	17	2197
  CCR5-1845	−	GGGGAGAAAAGACAUGA	17	2198
  CCR5-1846	−	GAAAUGACACUUUUCAU	17	2199
  CCR5-1847	−	AGAGUCAGCAGAACUGG	17	2200
  CCR5-1848	−	AGAAUCCCUAGUCUUCA	17	2201
  CCR5-1849	−	GUUUAGGUCAAGAAGAA	17	2202
  CCR5-1850	−	CUGAAUGCUUCUGACUU	17	2203
  CCR5-1851	−	GGGGUCUCCAGGAGGAG	17	2204
  CCR5-1852	−	CACACAUGAGAUCUAGG	17	2205
  CCR5-1853	−	CAUGAGAUCUAGGUGAG	17	2206
  CCR5-1854	−	CAUUUCAUGGGUUGUUG	17	2207
  CCR5-1855	−	UUUUUCUGUUCUGUCUC	17	2208
  CCR5-1856	+	UUCAUUUCAAAGGGAGGGAG	20	2209
  CCR5-1857	+	UCUCCAAUCUGCUUGAAGAC	20	2210
  CCR5-1858	+	UGCUAUUUUUCAUCAACAUA	20	2211
  CCR5-1859	+	UCGACACCGAAGCAGAGUUU	20	2212
  CCR5-1860	+	AUUUCAAAGGGAGGGAG	17	2213
  CCR5-1861	+	CCAAUCUGCUUGAAGAC	17	2214
  CCR5-1862	+	UAUUUUUCAUCAACAUA	17	2215
  CCR5-1863	+	ACACCGAAGCAGAGUUU	17	2216

• Table 2A provides exemplary targeting domains for knocking out the CCR5 gene selected according to the first tier parameters. The targeting domains bind within the first 500 bp of the coding sequence (e.g., within 500 bp downstream from the start codon) and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 2A
  1st Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-115	−	ACUAUGCUGCCGCCCAG	17	4343
  CCR5-121	−	UCCUCCUGACAAUCGAU	17	4344
  CCR5-116	−	CUAUGCUGCCGCCCAGU	17	4345
  CCR5-3	−	GCCGCCCAGUGGGACUU	17	4346
  CCR5-53	−	UUGACAGGGCUCUAUUUUAU	20	4347
  CCR5-75	−	UCACUAUGCUGCCGCCCAGU	20	4348

• Table 2B provides exemplary targeting domains for knocking out the CCR5 gene selected according to the second tier parameters. The targeting domains bind within the first 500 bp of the coding sequence (e.g., within 500 bp downstream from the start codon). It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 2B
  2nd Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-111	−	UCCUGAUAAACUGCAAA	17	4349
  CCR5-135	+	ACUUGUCACCACCCCAA	17	4350
  CCR5-4	+	GCAUAGUGAGCCCAGAA	17	4351
  CCR5-1864	−	CUUUUUAUUUAUGCACA	17	4352
  CCR5-118	−	UGUGUCAACUCUUGACA	17	4353
  CCR5-151	+	UUAAAGCAAACACAGCA	17	4354
  CCR5-132	+	ACAUUGAUUUUUUGGCA	17	4355
  CCR5-1865	−	ACCAGAUCUCAAAAAGA	17	4356
  CCR5-1866	−	CACAGGGUGGAACAAGA	17	4357
  CCR5-136	+	AGAAGGGGACAGUAAGA	17	4358
  CCR5-139	+	AGCAUAGUGAGCCCAGA	17	4359
  CCR5-5	+	GAAAAACAGGUCAGAGA	17	4360
  CCR5-123	−	UGCUUUAAAAGCCAGGA	17	4361
  CCR5-144	+	CAGUAAGAAGGAAAAAC	17	4362
  CCR5-148	+	UAUUUCCAAAGUCCCAC	17	4363
  CCR5-1867	−	ACUUUUUAUUUAUGCAC	17	4364
  CCR5-1	−	GCCUCCGCUCUACUCAC	17	4365
  CCR5-52	−	AUGUGUCAACUCUUGAC	17	4366
  CCR5-112	−	CAUCUACCUGCUCAACC	17	4367
  CCR5-10	−	GACAAUCGAUAGGUACC	17	4368
  CCR5-129	−	GUGUUUGCGUCUCUCCC	17	4369
  CCR5-122	−	UGUUUGCUUUAAAAGCC	17	4370
  CCR5-143	+	CAGCAUGGACGACAGCC	17	4371
  CCR5-131	+	ACAGGUCAGAGAUGGCC	17	4372
  CCR5-146	+	CCCAAAGGUGACCGUCC	17	4373
  CCR5-1868	+	CUGGUAAAGAUGAUUCC	17	4374
  CCR5-138	+	AGAUGGCCAGGUUGAGC	17	4375
  CCR5-8	+	GAGCGGAGGCAGGAGGC	17	4376
  CCR5-7	+	GUGAGUAGAGCGGAGGC	17	4377
  CCR5-64	+	CACAUUGAUUUUUUGGC	17	4378
  CCR5-110	−	UUUUGUGGGCAACAUGC	17	4379
  CCR5-1869	+	ACCUUCUUUUUGAGAUC	17	4380
  CCR5-6	+	GCCUUUUGCAGUUUAUC	17	4381
  CCR5-120	−	UUUAUAGGCUUCUUCUC	17	4382
  CCR5-14	+	GGUACCUAUCGAUUGUC	17	4383
  CCR5-113	−	UUCUUACUGUCCCCUUC	17	4384
  CCR5-145	+	CAUAGUGAGCCCAGAAG	17	4385
  CCR5-130	+	AACACCAGUGAGUAGAG	17	4386
  CCR5-65	+	AGUAGAGCGGAGGCAGG	17	4387
  CCR5-134	+	ACCUAUCGAUUGUCAGG	17	4388
  CCR5-137	+	AGAGCGGAGGCAGGAGG	17	4389
  CCR5-133	+	ACCAGUGAGUAGAGCGG	17	4390
  CCR5-1870	−	UUUAUUUAUGCACAGGG	17	4391
  CCR5-12	−	GACGGUCACCUUUGGGG	17	4392
  CCR5-149	+	UCCAAAGUCCCACUGGG	17	4393
  CCR5-127	−	AAGUGUGAUCACUUGGG	17	4394
  CCR5-128	−	UGUGAUCACUUGGGUGG	17	4395
  CCR5-150	+	UGCAGUUUAUCAGGAUG	17	4396
  CCR5-125	−	CAGGACGGUCACCUUUG	17	4397
  CCR5-2	−	GUUCAUCUUUGGUUUUG	17	4398
  CCR5-107	−	CAUCAAUUAUUAUACAU	17	4399
  CCR5-147	+	UAAUUGAUGUCAUAGAU	17	4400
  CCR5-119	−	ACAGGGCUCUAUUUUAU	17	4401
  CCR5-141	+	AUUUCCAAAGUCCCACU	17	4402
  CCR5-126	−	UGACAAGUGUGAUCACU	17	4403
  CCR5-1871	+	UGGUAAAGAUGAUUCCU	17	4404
  CCR5-114	−	UCUUACUGUCCCCUUCU	17	4405
  CCR5-109	−	UUCAUCUUUGGUUUUGU	17	4406
  CCR5-13	−	GACAAGUGUGAUCACUU	17	4407
  CCR5-11	−	GCCAGGACGGUCACCUU	17	4408
  CCR5-108	−	UCACUGGUGUUCAUCUU	17	4409
  CCR5-124	−	CCAGGACGGUCACCUUU	17	4410
  CCR5-9	+	GCUUCACAUUGAUUUUU	17	4411
  CCR5-70	−	UCAUCCUGAUAAACUGCAAA	20	4412
  CCR5-94	+	CACACUUGUCACCACCCCAA	20	4413
  CCR5-47	+	GCAGCAUAGUGAGCCCAGAA	20	4414
  CCR5-76	−	CAAUGUGUCAACUCUUGACA	20	4415
  CCR5-100	+	CUUUUAAAGCAAACACAGCA	20	4416
  CCR5-103	+	UUCACAUUGAUUUUUUGGCA	20	4417
  CCR5-1872	−	UUUACCAGAUCUCAAAAAGA	20	4418
  CCR5-1873	−	AUGCACAGGGUGGAACAAGA	20	4419
  CCR5-99	+	CCCAGAAGGGGACAGUAAGA	20	4420
  CCR5-46	+	GGCAGCAUAGUGAGCCCAGA	20	4421
  CCR5-89	+	AAGGAAAAACAGGUCAGAGA	20	4422
  CCR5-79	−	GUUUGCUUUAAAAGCCAGGA	20	4423
  CCR5-48	+	GGACAGUAAGAAGGAAAAAC	20	4424
  CCR5-104	+	UUGUAUUUCCAAAGUCCCAC	20	4425
  CCR5-66	−	CCUGCCUCCGCUCUACUCAC	20	4426
  CCR5-51	−	ACAAUGUGUCAACUCUUGAC	20	4427
  CCR5-71	−	UGACAUCUACCUGCUCAACC	20	4428
  CCR5-57	−	CCUGACAAUCGAUAGGUACC	20	4429
  CCR5-59	−	GCUGUGUUUGCGUCUCUCCC	20	4430
  CCR5-78	−	CUGUGUUUGCUUUAAAAGCC	20	4431
  CCR5-90	+	ACACAGCAUGGACGACAGCC	20	4432
  CCR5-87	+	AAAACAGGUCAGAGAUGGCC	20	4433
  CCR5-95	+	CACCCCAAAGGUGACCGUCC	20	4434
  CCR5-1874	+	GAUCUGGUAAAGAUGAUUCC	20	4435
  CCR5-96	+	CAGAGAUGGCCAGGUUGAGC	20	4436
  CCR5-50	+	GUAGAGCGGAGGCAGGAGGC	20	4437
  CCR5-98	+	CCAGUGAGUAGAGCGGAGGC	20	4438
  CCR5-63	+	CUUCACAUUGAUUUUUUGGC	20	4439
  CCR5-69	−	UGGUUUUGUGGGCAACAUGC	20	4440
  CCR5-1875	+	AAGACCUUCUUUUUGAGAUC	20	4441
  CCR5-62	+	UCAGCCUUUUGCAGUUUAUC	20	4442
  CCR5-77	−	UAUUUUAUAGGCUUCUUCUC	20	4443
  CCR5-60	+	CCAGGUACCUAUCGAUUGUC	20	4444
  CCR5-72	−	UCCUUCUUACUGUCCCCUUC	20	4445
  CCR5-97	+	CAGCAUAGUGAGCCCAGAAG	20	4446
  CCR5-74	−	CUCACUAUGCUGCCGCCCAG	20	4447
  CCR5-92	+	AUGAACACCAGUGAGUAGAG	20	4448
  CCR5-49	+	GUGAGUAGAGCGGAGGCAGG	20	4449
  CCR5-45	+	GGUACCUAUCGAUUGUCAGG	20	4450
  CCR5-91	+	AGUAGAGCGGAGGCAGGAGG	20	4451
  CCR5-88	+	AACACCAGUGAGUAGAGCGG	20	4452
  CCR5-1876	−	CUUUUUAUUUAUGCACAGGG	20	4453
  CCR5-83	−	CAGGACGGUCACCUUUGGGG	20	4454
  CCR5-93	+	AUUUCCAAAGUCCCACUGGG	20	4455
  CCR5-85	−	GACAAGUGUGAUCACUUGGG	20	4456
  CCR5-86	−	AAGUGUGAUCACUUGGGUGG	20	4457
  CCR5-106	+	UUUUGCAGUUUAUCAGGAUG	20	4458
  CCR5-82	−	AGCCAGGACGGUCACCUUUG	20	4459
  CCR5-41	−	GGUGUUCAUCUUUGGUUUUG	20	4460
  CCR5-67	−	UGACAUCAAUUAUUAUACAU	20	4461
  CCR5-101	+	UAAUAAUUGAUGUCAUAGAU	20	4462
  CCR5-55	−	UCAUCCUCCUGACAAUCGAU	20	4463
  CCR5-102	+	UGUAUUUCCAAAGUCCCACU	20	4464
  CCR5-84	−	UGGUGACAAGUGUGAUCACU	20	4465
  CCR5-1877	+	AUCUGGUAAAGAUGAUUCCU	20	4466
  CCR5-73	−	CCUUCUUACUGUCCCCUUCU	20	4467
  CCR5-42	−	GUGUUCAUCUUUGGUUUUGU	20	4468
  CCR5-58	−	GGUGACAAGUGUGAUCACUU	20	4469
  CCR5-43	−	GCUGCCGCCCAGUGGGACUU	20	4470
  CCR5-80	−	AAAGCCAGGACGGUCACCUU	20	4471
  CCR5-68	−	UACUCACUGGUGUUCAUCUU	20	4472
  CCR5-81	−	AAGCCAGGACGGUCACCUUU	20	4473
  CCR5-105	+	UUUGCUUCACAUUGAUUUUU	20	4474

• Table 2C provides exemplary targeting domains for knocking out the CCR5 gene selected according to the third tier parameters. The targeting domains fall in the coding sequence of the gene, downstream of the first 500 bp of coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon of the gene). It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 2C
  3rd Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO

  CCR5-793	+	GAACUUCUCCCCGACAA	17	4475
  CCR5-382	−	UGAGAAGAAGAGGCACA	17	4476
  CCR5-403	−	UCUGUGGGCUUGUGACA	17	4477
  CCR5-376	−	CCUGCCGCUGCUUGUCA	17	4478
  CCR5-1865	−	ACCAGAUCUCAAAAAGA	17	4479
  CCR5-802	+	GGAAGGUGUUCAGGAGA	17	4480
  CCR5-800	+	GCCAAAGAAUUCCUGGA	17	4481
  CCR5-805	+	AAAAUAAACAAUCAUGA	17	4482
  CCR5-794	+	GACAAAGGCAUAGAUGA	17	4483
  CCR5-810	+	AAUUGAUACUGACUGUA	17	4484
  CCR5-804	+	AGAAGGACAAUGUUGUA	17	4485
  CCR5-388	−	AUUGCAGUAGCUCUAAC	17	4486
  CCR5-397	−	GUUUACACCCGAUCCAC	17	4487
  CCR5-381	−	AUGAGAAGAAGAGGCAC	17	4488
  CCR5-799	+	UCAGGCCAAAGAAUUCC	17	4489
  CCR5-1868	+	CUGGUAAAGAUGAUUCC	17	4490
  CCR5-386	−	UCUCCUGAACACCUUCC	17	4491
  CCR5-400	−	CCGAUCCACUGGGGAGC	17	4492
  CCR5-808	+	CCAUGACAAGCAGCGGC	17	4493
  CCR5-375	−	GAUAGUCAUCUUGGGGC	17	4494
  CCR5-406	−	CACGGACUCAAGUGGGC	17	4495
  CCR5-390	−	GUUGGACCAAGCUAUGC	17	4496
  CCR5-811	+	UGGAAAAUGAGAGCUGC	17	4497
  CCR5-789	+	GCUCGGGAGCCUCUUGC	17	4498
  CCR5-1869	+	ACCUUCUUUUUGAGAUC	17	4499
  CCR5-786	+	CUGCUCCCCAGUGGAUC	17	4500
  CCR5-378	−	AUGGUCAUCUGCUACUC	17	4501
  CCR5-788	+	ACUGAGCUUGCUCGCUC	17	4502
  CCR5-809	+	UGACUAUCUUUAAUGUC	17	4503
  CCR5-394	−	UCAUCUAUGCCUUUGUC	17	4504
  CCR5-371	−	ACAGUCAGUAUCAAUUC	17	4505
  CCR5-798	+	AGCUACUGCAAUUAUUC	17	4506
  CCR5-384	−	UUGUUUAUUUUCUCUUC	17	4507
  CCR5-801	+	AUUCCUGGAAGGUGUUC	17	4508
  CCR5-396	−	UUCUAUUUUCCAGCAAG	17	4509
  CCR5-404	−	UGUGACACGGACUCAAG	17	4510
  CCR5-380	−	GUCGAAAUGAGAAGAAG	17	4511
  CCR5-792	+	UUUGGAAGAAGACUAAG	17	4512
  CCR5-784	+	UAUUUCCUGCUCCCCAG	17	4513
  CCR5-807	+	AUGACCAUGACAAGCAG	17	4514
  CCR5-395	−	CAUCUAUGCCUUUGUCG	17	4515
  CCR5-796	+	CAAAGGCAUAGAUGAUG	17	4516
  CCR5-399	−	UUACACCCGAUCCACUG	17	4517
  CCR5-401	−	GGAGCAGGAAAUAUCUG	17	4518
  CCR5-383	−	AGAGGCACAGGGCUGUG	17	4519
  CCR5-374	−	UAAAGAUAGUCAUCUUG	17	4520
  CCR5-785	+	CCUGCUCCCCAGUGGAU	17	4521
  CCR5-795	+	ACAAAGGCAUAGAUGAU	17	4522
  CCR5-398	−	UUUACACCCGAUCCACU	17	4523
  CCR5-377	−	CAUGGUCAUCUGCUACU	17	4524
  CCR5-1871	+	UGGUAAAGAUGAUUCCU	17	4525
  CCR5-797	+	CUGUCACCUGCAUAGCU	17	4526
  CCR5-787	+	AACUGAGCUUGCUCGCU	17	4527
  CCR5-372	−	AUUAAAGAUAGUCAUCU	17	4528
  CCR5-391	−	CAGGUGACAGAGACUCU	17	4529
  CCR5-385	−	UGUUUAUUUUCUCUUCU	17	4530
  CCR5-405	−	GUGACACGGACUCAAGU	17	4531
  CCR5-389	−	CAGUAGCUCUAACAGGU	17	4532
  CCR5-402	−	GAGCAGGAAAUAUCUGU	17	4533
  CCR5-803	+	GAGAAGGACAAUGUUGU	17	4534
  CCR5-393	−	AUCAUCUAUGCCUUUGU	17	4535
  CCR5-379	−	UCCUAAAAACUCUGCUU	17	4536
  CCR5-373	−	UUAAAGAUAGUCAUCUU	17	4537
  CCR5-392	−	AGGUGACAGAGACUCUU	17	4538
  CCR5-387	−	ACCUUCCAGGAAUUCUU	17	4539
  CCR5-790	+	GCAUUUGCAGAAGCGUU	17	4540
  CCR5-791	+	GUUUGGCAAUGUGCUUU	17	4541
  CCR5-806	+	ACCGAAGCAGAGUUUUU	17	4542
  CCR5-682	+	UCUGAACUUCUCCCCGACAA	20	4543
  CCR5-163	−	AAAUGAGAAGAAGAGGCACA	20	4544
  CCR5-184	−	AUAUCUGUGGGCUUGUGACA	20	4545
  CCR5-157	−	GGUCCUGCCGCUGCUUGUCA	20	4546
  CCR5-1872	−	UUUACCAGAUCUCAAAAAGA	20	4547
  CCR5-691	+	CCUGGAAGGUGUUCAGGAGA	20	4548
  CCR5-689	+	CAGGCCAAAGAAUUCCUGGA	20	4549
  CCR5-694	+	GAGAAAAUAAACAAUCAUGA	20	4550
  CCR5-683	+	CCCGACAAAGGCAUAGAUGA	20	4551
  CCR5-699	+	CAGAAUUGAUACUGACUGUA	20	4552
  CCR5-693	+	AGGAGAAGGACAAUGUUGUA	20	4553
  CCR5-169	−	AUAAUUGCAGUAGCUCUAAC	20	4554
  CCR5-178	−	UCAGUUUACACCCGAUCCAC	20	4555
  CCR5-162	−	GAAAUGAGAAGAAGAGGCAC	20	4556
  CCR5-688	+	UAUUCAGGCCAAAGAAUUCC	20	4557
  CCR5-1874	+	GAUCUGGUAAAGAUGAUUCC	20	4558
  CCR5-167	−	CCUUCUCCUGAACACCUUCC	20	4559
  CCR5-181	−	CACCCGAUCCACUGGGGAGC	20	4560
  CCR5-697	+	UGACCAUGACAAGCAGCGGC	20	4561
  CCR5-156	−	AAAGAUAGUCAUCUUGGGGC	20	4562
  CCR5-187	−	UGACACGGACUCAAGUGGGC	20	4563
  CCR5-171	−	CAGGUUGGACCAAGCUAUGC	20	4564
  CCR5-700	+	GUAUGGAAAAUGAGAGCUGC	20	4565
  CCR5-678	+	CUCGCUCGGGAGCCUCUUGC	20	4566
  CCR5-1875	+	AAGACCUUCUUUUUGAGAUC	20	4567
  CCR5-675	+	UUCCUGCUCCCCAGUGGAUC	20	4568
  CCR5-159	−	GUCAUGGUCAUCUGCUACUC	20	4569
  CCR5-677	+	UAAACUGAGCUUGCUCGCUC	20	4570
  CCR5-698	+	AGAUGACUAUCUUUAAUGUC	20	4571
  CCR5-175	−	CCAUCAUCUAUGCCUUUGUC	20	4572
  CCR5-152	−	CAUACAGUCAGUAUCAAUUC	20	4573
  CCR5-687	+	UAGAGCUACUGCAAUUAUUC	20	4574
  CCR5-165	−	UGAUUGUUUAUUUUCUCUUC	20	4575
  CCR5-690	+	AGAAUUCCUGGAAGGUGUUC	20	4576
  CCR5-177	−	CUGUUCUAUUUUCCAGCAAG	20	4577
  CCR5-185	−	GCUUGUGACACGGACUCAAG	20	4578
  CCR5-161	−	GGUGUCGAAAUGAGAAGAAG	20	4579
  CCR5-681	+	GCUUUUGGAAGAAGACUAAG	20	4580
  CCR5-673	+	AGAUAUUUCCUGCUCCCCAG	20	4581
  CCR5-696	+	CAGAUGACCAUGACAAGCAG	20	4582
  CCR5-176	−	CAUCAUCUAUGCCUUUGUCG	20	4583
  CCR5-685	+	CGACAAAGGCAUAGAUGAUG	20	4584
  CCR5-180	−	AGUUUACACCCGAUCCACUG	20	4585
  CCR5-182	−	UGGGGAGCAGGAAAUAUCUG	20	4586
  CCR5-164	−	AGAAGAGGCACAGGGCUGUG	20	4587
  CCR5-155	−	CAUUAAAGAUAGUCAUCUUG	20	4588
  CCR5-674	+	UUUCCUGCUCCCCAGUGGAU	20	4589
  CCR5-684	+	CCGACAAAGGCAUAGAUGAU	20	4590
  CCR5-179	−	CAGUUUACACCCGAUCCACU	20	4591
  CCR5-158	−	UGUCAUGGUCAUCUGCUACU	20	4592
  CCR5-1877	+	AUCUGGUAAAGAUGAUUCCU	20	4593
  CCR5-686	+	UCUCUGUCACCUGCAUAGCU	20	4594
  CCR5-676	+	GUAAACUGAGCUUGCUCGCU	20	4595
  CCR5-153	−	GACAUUAAAGAUAGUCAUCU	20	4596
  CCR5-172	−	AUGCAGGUGACAGAGACUCU	20	4597
  CCR5-166	−	GAUUGUUUAUUUUCUCUUCU	20	4598
  CCR5-186	−	CUUGUGACACGGACUCAAGU	20	4599
  CCR5-170	−	UUGCAGUAGCUCUAACAGGU	20	4600
  CCR5-183	−	GGGGAGCAGGAAAUAUCUGU	20	4601
  CCR5-692	+	CAGGAGAAGGACAAUGUUGU	20	4602
  CCR5-174	−	CCCAUCAUCUAUGCCUUUGU	20	4603
  CCR5-160	−	GAAUCCUAAAAACUCUGCUU	20	4604
  CCR5-154	−	ACAUUAAAGAUAGUCAUCUU	20	4605
  CCR5-173	−	UGCAGGUGACAGAGACUCUU	20	4606
  CCR5-168	−	AACACCUUCCAGGAAUUCUU	20	4607
  CCR5-679	+	ACAGCAUUUGCAGAAGCGUU	20	4608
  CCR5-680	+	AGCGUUUGGCAAUGUGCUUU	20	4609
  CCR5-695	+	GACACCGAAGCAGAGUUUUU	20	4610

• Table 3A provides exemplary targeting domains for knocking out the CCR5 gene selected according to the first tier parameters. The targeting domains bind within the first 500 bp of the coding sequence (e.g., within 500 bp downstream from the start codon), have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 3A
  1st Tier

  gRNA	DNA		Target Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO

  CCR5-1878	+	AUAAAAUAGAGCCCUGUC	18	4611
  CCR5-1879	+	UAUAAAAUAGAGCCCUGUC	19	4612
  CCR5-862	+	CUAUAAAAUAGAGCCCUGUC	20	4613
  CCR5-1880	+	CCUAUAAAAUAGAGCCCUGUC	21	4614
  CCR5-1881	+	GCCUAUAAAAUAGAGCCCUGUC	22	4615
  CCR5-1882	+	AGCCUAUAAAAUAGAGCCCUGUC	23	4616
  CCR5-1883	+	AAGCCUAUAAAAUAGAGCCCUGUC	24	4617
  CCR5-1884	+	UUUGCAGUUUAUCAGGAU	18	4618
  CCR5-1885	+	UUUUGCAGUUUAUCAGGAU	19	4619
  CCR5-876	+	CUUUUGCAGUUUAUCAGGAU	20	4620
  CCR5-1886	−	GGUGACAAGUGUGAUCAC	18	4621
  CCR5-1887	−	UGGUGACAAGUGUGAUCAC	19	4622
  CCR5-829	−	GUGGUGACAAGUGUGAUCAC	20	4623
  CCR5-1888	−	GGUGGUGACAAGUGUGAUCAC	21	4624
  CCR5-1889	−	GGGUGGUGACAAGUGUGAUCAC	22	4625
  CCR5-1890	−	GGGGUGGUGACAAGUGUGAUCAC	23	4626
  CCR5-1891	−	UGGGGUGGUGACAAGUGUGAUCAC	24	4627
  CCR5-1892	−	UUAUGCACAGGGUGGAACAAG	21	4628
  CCR5-1893	−	UUUAUGCACAGGGUGGAACAAG	22	4629
  CCR5-1894	−	AUUUAUGCACAGGGUGGAACAAG	23	4630
  CCR5-1895	−	UAUUUAUGCACAGGGUGGAACAAG	24	4631

• Table 3B provides exemplary targeting domains for knocking out the CCR5 gene selected according to the second tier parameters. The targeting domains bind within the first 500 bp of the coding sequence (e.g., with 500 bp downstream from the start codon) and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 3B
  2nd Tier

  gRNA	DNA		Target Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO

  CCR5-1896	+	AACCAAAGAUGAACACCA	18	4632
  CCR5-1897	+	AAACCAAAGAUGAACACCA	19	4633
  CCR5-878	+	AAAACCAAAGAUGAACACCA	20	4634
  CCR5-1898	+	CAAAACCAAAGAUGAACACCA	21	4635
  CCR5-1899	+	ACAAAACCAAAGAUGAACACCA	22	4636
  CCR5-1900	+	CACAAAACCAAAGAUGAACACCA	23	4637
  CCR5-1901	+	CCACAAAACCAAAGAUGAACACCA	24	4638
  CCR5-1902	+	GUACCUAUCGAUUGUCAG	18	4639
  CCR5-1903	+	GGUACCUAUCGAUUGUCAG	19	4640
  CCR5-855	+	AGGUACCUAUCGAUUGUCAG	20	4641
  CCR5-1904	+	CAGGUACCUAUCGAUUGUCAG	21	4642
  CCR5-1905	+	CCAGGUACCUAUCGAUUGUCAG	22	4643
  CCR5-1906	+	GCCAGGUACCUAUCGAUUGUCAG	23	4644
  CCR5-1907	+	AGCCAGGUACCUAUCGAUUGUCAG	24	4645
  CCR5-1908	+	CCUUUUGCAGUUUAUCAGGAU	21	4646
  CCR5-1909	+	GCCUUUUGCAGUUUAUCAGGAU	22	4647
  CCR5-1910	+	AGCCUUUUGCAGUUUAUCAGGAU	23	4648
  CCR5-1911	+	CAGCCUUUUGCAGUUUAUCAGGAU	24	4649
  CCR5-1912	+	CAGCCUUUUGCAGUUUAU	18	4650
  CCR5-1913	+	UCAGCCUUUUGCAGUUUAU	19	4651
  CCR5-874	+	UUCAGCCUUUUGCAGUUUAU	20	4652
  CCR5-1914	+	CUUCAGCCUUUUGCAGUUUAU	21	4653
  CCR5-1915	+	UCUUCAGCCUUUUGCAGUUUAU	22	4654
  CCR5-1916	+	CUCUUCAGCCUUUUGCAGUUUAU	23	4655
  CCR5-1917	+	GCUCUUCAGCCUUUUGCAGUUUAU	24	4656
  CCR5-1918	−	UGUGUUUGCGUCUCUCCC	18	4657
  CCR5-1919	−	CUGUGUUUGCGUCUCUCCC	19	4658
  CCR5-59	−	GCUGUGUUUGCGUCUCUCCC	20	4659
  CCR5-1920	−	GGCUGUGUUUGCGUCUCUCCC	21	4660
  CCR5-1921	−	UGGCUGUGUUUGCGUCUCUCCC	22	4661
  CCR5-1922	−	GUGGCUGUGUUUGCGUCUCUCCC	23	4662
  CCR5-1923	−	GGUGGCUGUGUUUGCGUCUCUCCC	24	4663
  CCR5-1924	−	UUUUAUAGGCUUCUUCUC	18	4664
  CCR5-1925	−	AUUUUAUAGGCUUCUUCUC	19	4665
  CCR5-77	−	UAUUUUAUAGGCUUCUUCUC	20	4666
  CCR5-1926	−	CUAUUUUAUAGGCUUCUUCUC	21	4667
  CCR5-1927	−	UCUAUUUUAUAGGCUUCUUCUC	22	4668
  CCR5-1928	−	CUCUAUUUUAUAGGCUUCUUCUC	23	4669
  CCR5-1929	−	GCUCUAUUUUAUAGGCUUCUUCUC	24	4670
  CCR5-1930	−	UGCACAGGGUGGAACAAG	18	4671
  CCR5-1931	−	AUGCACAGGGUGGAACAAG	19	4672
  CCR5-1932	−	UAUGCACAGGGUGGAACAAG	20	4673
  CCR5-1933	−	AGCCAGGACGGUCACCUU	18	4674
  CCR5-1934	−	AAGCCAGGACGGUCACCUU	19	4675
  CCR5-80	−	AAAGCCAGGACGGUCACCUU	20	4676
  CCR5-1935	−	AAAAGCCAGGACGGUCACCUU	21	4677
  CCR5-1936	−	UAAAAGCCAGGACGGUCACCUU	22	4678
  CCR5-1937	−	UUAAAAGCCAGGACGGUCACCUU	23	4679
  CCR5-1938	−	UUUAAAAGCCAGGACGGUCACCUU	24	4680

• Table 3C provides exemplary targeting domains for knocking out the CCR5 gene selected according to the third tier parameters. The targeting domains bind within the first 500 bp of the coding sequence (e.g., with 500 bp downstream from the start codon) and PAM is NNGRRV. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 3C
  3rd Tier

  gRNA	DNA		Target Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO

  CCR5-2255	+	GAUAUUUCCUGCUCCCCA	18	4681
  CCR5-2256	+	AGAUAUUUCCUGCUCCCCA	19	4682
  CCR5-1611	+	CAGAUAUUUCCUGCUCCCCA	20	4683
  CCR5-2257	+	ACAGAUAUUUCCUGCUCCCCA	21	4684
  CCR5-2258	+	CACAGAUAUUUCCUGCUCCCCA	22	4685
  CCR5-2259	+	CCACAGAUAUUUCCUGCUCCCCA	23	4686
  CCR5-2260	+	CCCACAGAUAUUUCCUGCUCCCCA	24	4687
  CCR5-2261	+	CUGCAAUUAUUCAGGCCA	18	4688
  CCR5-2262	+	ACUGCAAUUAUUCAGGCCA	19	4689
  CCR5-1630	+	UACUGCAAUUAUUCAGGCCA	20	4690
  CCR5-2263	+	CUACUGCAAUUAUUCAGGCCA	21	4691
  CCR5-2264	+	GCUACUGCAAUUAUUCAGGCCA	22	4692
  CCR5-2265	+	AGCUACUGCAAUUAUUCAGGCCA	23	4693
  CCR5-2266	+	GAGCUACUGCAAUUAUUCAGGCCA	24	4694
  CCR5-2267	+	UUCCUGCUCCCCAGUGGA	18	4695
  CCR5-2268	+	UUUCCUGCUCCCCAGUGGA	19	4696
  CCR5-1612	+	AUUUCCUGCUCCCCAGUGGA	20	4697
  CCR5-2269	+	UAUUUCCUGCUCCCCAGUGGA	21	4698
  CCR5-2270	+	AUAUUUCCUGCUCCCCAGUGGA	22	4699
  CCR5-2271	+	GAUAUUUCCUGCUCCCCAGUGGA	23	4700
  CCR5-2272	+	AGAUAUUUCCUGCUCCCCAGUGGA	24	4701
  CCR5-2273	+	CGACAAAGGCAUAGAUGA	18	4702
  CCR5-2274	+	CCGACAAAGGCAUAGAUGA	19	4703
  CCR5-683	+	CCCGACAAAGGCAUAGAUGA	20	4704
  CCR5-2275	+	CCCCGACAAAGGCAUAGAUGA	21	4705
  CCR5-2276	+	UCCCCGACAAAGGCAUAGAUGA	22	4706
  CCR5-2277	+	CUCCCCGACAAAGGCAUAGAUGA	23	4707
  CCR5-2278	+	UCUCCCCGACAAAGGCAUAGAUGA	24	4708
  CCR5-2279	+	GCAGCAGUGCGUCAUCCC	18	4709
  CCR5-2280	+	UGCAGCAGUGCGUCAUCCC	19	4710
  CCR5-1628	+	AUGCAGCAGUGCGUCAUCCC	20	4711
  CCR5-2281	+	GAUGCAGCAGUGCGUCAUCCC	21	4712
  CCR5-2282	+	UGAUGCAGCAGUGCGUCAUCCC	22	4713
  CCR5-2283	+	UUGAUGCAGCAGUGCGUCAUCCC	23	4714
  CCR5-2284	+	GUUGAUGCAGCAGUGCGUCAUCCC	24	4715
  CCR5-2285	+	GCAGAGUUUUUAGGAUUC	18	4716
  CCR5-2286	+	AGCAGAGUUUUUAGGAUUC	19	4717
  CCR5-1647	+	AAGCAGAGUUUUUAGGAUUC	20	4718
  CCR5-2287	+	GAAGCAGAGUUUUUAGGAUUC	21	4719
  CCR5-2288	+	CGAAGCAGAGUUUUUAGGAUUC	22	4720
  CCR5-2289	+	CCGAAGCAGAGUUUUUAGGAUUC	23	4721
  CCR5-2290	+	ACCGAAGCAGAGUUUUUAGGAUUC	24	4722
  CCR5-2291	+	AAUGUCUGGAAAUUCUUC	18	4723
  CCR5-2292	+	UAAUGUCUGGAAAUUCUUC	19	4724
  CCR5-1651	+	UUAAUGUCUGGAAAUUCUUC	20	4725
  CCR5-2293	+	UUUAAUGUCUGGAAAUUCUUC	21	4726
  CCR5-2294	+	CUUUAAUGUCUGGAAAUUCUUC	22	4727
  CCR5-2295	+	UCUUUAAUGUCUGGAAAUUCUUC	23	4728
  CCR5-2296	+	AUCUUUAAUGUCUGGAAAUUCUUC	24	4729
  CCR5-2297	+	CUCAUUUCGACACCGAAG	18	4730
  CCR5-2298	+	UCUCAUUUCGACACCGAAG	19	4731
  CCR5-1645	+	UUCUCAUUUCGACACCGAAG	20	4732
  CCR5-2299	+	CUUCUCAUUUCGACACCGAAG	21	4733
  CCR5-2300	+	UCUUCUCAUUUCGACACCGAAG	22	4734
  CCR5-2301	+	UUCUUCUCAUUUCGACACCGAAG	23	4735
  CCR5-2302	+	CUUCUUCUCAUUUCGACACCGAAG	24	4736
  CCR5-2303	+	ACACCGAAGCAGAGUUUU	18	4737
  CCR5-2304	+	GACACCGAAGCAGAGUUUU	19	4738
  CCR5-1646	+	CGACACCGAAGCAGAGUUUU	20	4739
  CCR5-2305	+	UCGACACCGAAGCAGAGUUUU	21	4740
  CCR5-2306	+	UUCGACACCGAAGCAGAGUUUU	22	4741
  CCR5-2307	+	UUUCGACACCGAAGCAGAGUUUU	23	4742
  CCR5-2308	+	AUUUCGACACCGAAGCAGAGUUUU	24	4743
  CCR5-2309	−	UUCUCCUGAACACCUUCC	18	4744
  CCR5-2310	−	CUUCUCCUGAACACCUUCC	19	4745
  CCR5-167	−	CCUUCUCCUGAACACCUUCC	20	4746
  CCR5-2311	−	UCCUUCUCCUGAACACCUUCC	21	4747
  CCR5-2312	−	GUCCUUCUCCUGAACACCUUCC	22	4748
  CCR5-2313	−	UGUCCUUCUCCUGAACACCUUCC	23	4749
  CCR5-2314	−	UUGUCCUUCUCCUGAACACCUUCC	24	4750
  CCR5-2315	−	UUCCAGGAAUUCUUUGGC	18	4751
  CCR5-2316	−	CUUCCAGGAAUUCUUUGGC	19	4752
  CCR5-941	−	CCUUCCAGGAAUUCUUUGGC	20	4753
  CCR5-2317	−	ACCUUCCAGGAAUUCUUUGGC	21	4754
  CCR5-2318	−	CACCUUCCAGGAAUUCUUUGGC	22	4755
  CCR5-2319	−	ACACCUUCCAGGAAUUCUUUGGC	23	4756
  CCR5-2320	−	AACACCUUCCAGGAAUUCUUUGGC	24	4757
  CCR5-2321	−	CAUGGUCAUCUGCUACUC	18	4758
  CCR5-2322	−	UCAUGGUCAUCUGCUACUC	19	4759
  CCR5-159	−	GUCAUGGUCAUCUGCUACUC	20	4760
  CCR5-2323	−	UGUCAUGGUCAUCUGCUACUC	21	4761
  CCR5-2324	−	UUGUCAUGGUCAUCUGCUACUC	22	4762
  CCR5-2325	−	CUUGUCAUGGUCAUCUGCUACUC	23	4763
  CCR5-2326	−	GCUUGUCAUGGUCAUCUGCUACUC	24	4764
  CCR5-2327	−	AGUCAGUAUCAAUUCUGG	18	4765
  CCR5-2328	−	CAGUCAGUAUCAAUUCUGG	19	4766
  CCR5-924	−	ACAGUCAGUAUCAAUUCUGG	20	4767
  CCR5-2329	−	UACAGUCAGUAUCAAUUCUGG	21	4768
  CCR5-2330	−	AUACAGUCAGUAUCAAUUCUGG	22	4769
  CCR5-2331	−	CAUACAGUCAGUAUCAAUUCUGG	23	4770
  CCR5-2332	−	CCAUACAGUCAGUAUCAAUUCUGG	24	4771
  CCR5-2333	−	GCAGGUGACAGAGACUCU	18	4772
  CCR5-2334	−	UGCAGGUGACAGAGACUCU	19	4773
  CCR5-172	−	AUGCAGGUGACAGAGACUCU	20	4774
  CCR5-2335	−	UAUGCAGGUGACAGAGACUCU	21	4775
  CCR5-2336	−	CUAUGCAGGUGACAGAGACUCU	22	4776
  CCR5-2337	−	GCUAUGCAGGUGACAGAGACUCU	23	4777
  CCR5-2338	−	AGCUAUGCAGGUGACAGAGACUCU	24	4778

• Table 3D provides exemplary targeting domains for knocking out the CCR5 gene selected according to the fourth tier parameters. The targeting domains fall in the coding sequence of the gene, downstream of the first 500 bp of coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon of the gene.) and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 3D
  3rd Tier

  gRNA	DNA		Target Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO

  CCR5-1939	+	GAGAAGAAGCCUAUAAAA	18	4779
  CCR5-1940	+	AGAGAAGAAGCCUAUAAAA	19	4780
  CCR5-861	+	CAGAGAAGAAGCCUAUAAAA	20	4781
  CCR5-1941	+	CCAGAGAAGAAGCCUAUAAAA	21	4782
  CCR5-1942	+	UCCAGAGAAGAAGCCUAUAAAA	22	4783
  CCR5-1943	+	UUCCAGAGAAGAAGCCUAUAAAA	23	4784
  CCR5-1944	+	AUUCCAGAGAAGAAGCCUAUAAAA	24	4785
  CCR5-1945	+	AGCAUAGUGAGCCCAGAA	18	4786
  CCR5-1946	+	CAGCAUAGUGAGCCCAGAA	19	4787
  CCR5-47	+	GCAGCAUAGUGAGCCCAGAA	20	4788
  CCR5-1947	+	GGCAGCAUAGUGAGCCCAGAA	21	4789
  CCR5-1948	+	CGGCAGCAUAGUGAGCCCAGAA	22	4790
  CCR5-1949	+	GCGGCAGCAUAGUGAGCCCAGAA	23	4791
  CCR5-1950	+	GGCGGCAGCAUAGUGAGCCCAGAA	24	4792
  CCR5-1951	+	UGUAUUUCCAAAGUCCCA	18	4793
  CCR5-1952	+	UUGUAUUUCCAAAGUCCCA	19	4794
  CCR5-863	+	AUUGUAUUUCCAAAGUCCCA	20	4795
  CCR5-1953	+	CAUUGUAUUUCCAAAGUCCCA	21	4796
  CCR5-1954	+	ACAUUGUAUUUCCAAAGUCCCA	22	4797
  CCR5-1955	+	CACAUUGUAUUUCCAAAGUCCCA	23	4798
  CCR5-1956	+	ACACAUUGUAUUUCCAAAGUCCCA	24	4799
  CCR5-1957	+	AUGAUGAAGAAGAUUCCA	18	4800
  CCR5-1958	+	GAUGAUGAAGAAGAUUCCA	19	4801
  CCR5-859	+	GGAUGAUGAAGAAGAUUCCA	20	4802
  CCR5-1959	+	AGGAUGAUGAAGAAGAUUCCA	21	4803
  CCR5-1960	+	GAGGAUGAUGAAGAAGAUUCCA	22	4804
  CCR5-1961	+	GGAGGAUGAUGAAGAAGAUUCCA	23	4805
  CCR5-1962	+	AGGAGGAUGAUGAAGAAGAUUCCA	24	4806
  CCR5-1963	+	CAGAAGGGGACAGUAAGA	18	4807
  CCR5-1964	+	CCAGAAGGGGACAGUAAGA	19	4808
  CCR5-99	+	CCCAGAAGGGGACAGUAAGA	20	4809
  CCR5-1965	+	GCCCAGAAGGGGACAGUAAGA	21	4810
  CCR5-1966	+	AGCCCAGAAGGGGACAGUAAGA	22	4811
  CCR5-1967	+	GAGCCCAGAAGGGGACAGUAAGA	23	4812
  CCR5-1968	+	UGAGCCCAGAAGGGGACAGUAAGA	24	4813
  CCR5-1969	+	CAGCAUAGUGAGCCCAGA	18	4814
  CCR5-1970	+	GCAGCAUAGUGAGCCCAGA	19	4815
  CCR5-46	+	GGCAGCAUAGUGAGCCCAGA	20	4816
  CCR5-1971	+	CGGCAGCAUAGUGAGCCCAGA	21	4817
  CCR5-1972	+	GCGGCAGCAUAGUGAGCCCAGA	22	4818
  CCR5-1973	+	GGCGGCAGCAUAGUGAGCCCAGA	23	4819
  CCR5-1974	+	GGGCGGCAGCAUAGUGAGCCCAGA	24	4820
  CCR5-1975	+	AAUAAUUGAUGUCAUAGA	18	4821
  CCR5-1976	+	UAAUAAUUGAUGUCAUAGA	19	4822
  CCR5-886	+	AUAAUAAUUGAUGUCAUAGA	20	4823
  CCR5-1977	+	UAUAAUAAUUGAUGUCAUAGA	21	4824
  CCR5-1978	+	GUAUAAUAAUUGAUGUCAUAGA	22	4825
  CCR5-1979	+	UGUAUAAUAAUUGAUGUCAUAGA	23	4826
  CCR5-1980	+	AUGUAUAAUAAUUGAUGUCAUAGA	24	4827
  CCR5-1981	+	UGAACACCAGUGAGUAGA	18	4828
  CCR5-1982	+	AUGAACACCAGUGAGUAGA	19	4829
  CCR5-880	+	GAUGAACACCAGUGAGUAGA	20	4830
  CCR5-1983	+	AGAUGAACACCAGUGAGUAGA	21	4831
  CCR5-1984	+	AAGAUGAACACCAGUGAGUAGA	22	4832
  CCR5-1985	+	AAAGAUGAACACCAGUGAGUAGA	23	4833
  CCR5-1986	+	CAAAGAUGAACACCAGUGAGUAGA	24	4834
  CCR5-1987	+	CCACUGGGCGGCAGCAUA	18	4835
  CCR5-1988	+	CCCACUGGGCGGCAGCAUA	19	4836
  CCR5-864	+	UCCCACUGGGCGGCAGCAUA	20	4837
  CCR5-1989	+	GUCCCACUGGGCGGCAGCAUA	21	4838
  CCR5-1990	+	AGUCCCACUGGGCGGCAGCAUA	22	4839
  CCR5-1991	+	AAGUCCCACUGGGCGGCAGCAUA	23	4840
  CCR5-1992	+	AAAGUCCCACUGGGCGGCAGCAUA	24	4841
  CCR5-1993	+	GCGGCAGCAUAGUGAGCC	18	4842
  CCR5-1994	+	GGCGGCAGCAUAGUGAGCC	19	4843
  CCR5-865	+	GGGCGGCAGCAUAGUGAGCC	20	4844
  CCR5-1995	+	UGGGCGGCAGCAUAGUGAGCC	21	4845
  CCR5-1996	+	CUGGGCGGCAGCAUAGUGAGCC	22	4846
  CCR5-1997	+	ACUGGGCGGCAGCAUAGUGAGCC	23	4847
  CCR5-1998	+	CACUGGGCGGCAGCAUAGUGAGCC	24	4848
  CCR5-1999	+	UCUGGUAAAGAUGAUUCC	18	4849
  CCR5-2000	+	AUCUGGUAAAGAUGAUUCC	19	4850
  CCR5-1874	+	GAUCUGGUAAAGAUGAUUCC	20	4851
  CCR5-2001	+	AGAUCUGGUAAAGAUGAUUCC	21	4852
  CCR5-2002	+	GAGAUCUGGUAAAGAUGAUUCC	22	4853
  CCR5-2003	+	UGAGAUCUGGUAAAGAUGAUUCC	23	4854
  CCR5-2004	+	UUGAGAUCUGGUAAAGAUGAUUCC	24	4855
  CCR5-2005	+	UUUUAAAGCAAACACAGC	18	4856
  CCR5-2006	+	CUUUUAAAGCAAACACAGC	19	4857
  CCR5-852	+	GCUUUUAAAGCAAACACAGC	20	4858
  CCR5-2007	+	GGCUUUUAAAGCAAACACAGC	21	4859
  CCR5-2008	+	UGGCUUUUAAAGCAAACACAGC	22	4860
  CCR5-2009	+	CUGGCUUUUAAAGCAAACACAGC	23	4861
  CCR5-2010	+	CCUGGCUUUUAAAGCAAACACAGC	24	4862
  CCR5-2011	+	AGUGAGUAGAGCGGAGGC	18	4863
  CCR5-2012	+	CAGUGAGUAGAGCGGAGGC	19	4864
  CCR5-98	+	CCAGUGAGUAGAGCGGAGGC	20	4865
  CCR5-2013	+	ACCAGUGAGUAGAGCGGAGGC	21	4866
  CCR5-2014	+	CACCAGUGAGUAGAGCGGAGGC	22	4867
  CCR5-2015	+	ACACCAGUGAGUAGAGCGGAGGC	23	4868
  CCR5-2016	+	AACACCAGUGAGUAGAGCGGAGGC	24	4869
  CCR5-2017	+	AGGUACCUAUCGAUUGUC	18	4870
  CCR5-2018	+	CAGGUACCUAUCGAUUGUC	19	4871
  CCR5-60	+	CCAGGUACCUAUCGAUUGUC	20	4872
  CCR5-2019	+	GCCAGGUACCUAUCGAUUGUC	21	4873
  CCR5-2020	+	AGCCAGGUACCUAUCGAUUGUC	22	4874
  CCR5-2021	+	CAGCCAGGUACCUAUCGAUUGUC	23	4875
  CCR5-2022	+	ACAGCCAGGUACCUAUCGAUUGUC	24	4876
  CCR5-2023	+	GGAUGAUGAAGAAGAUUC	18	4877
  CCR5-2024	+	AGGAUGAUGAAGAAGAUUC	19	4878
  CCR5-858	+	GAGGAUGAUGAAGAAGAUUC	20	4879
  CCR5-2025	+	GGAGGAUGAUGAAGAAGAUUC	21	4880
  CCR5-2026	+	AGGAGGAUGAUGAAGAAGAUUC	22	4881
  CCR5-2027	+	CAGGAGGAUGAUGAAGAAGAUUC	23	4882
  CCR5-2028	+	UCAGGAGGAUGAUGAAGAAGAUUC	24	4883
  CCR5-2029	+	AUCUGGUAAAGAUGAUUC	18	4884
  CCR5-2030	+	GAUCUGGUAAAGAUGAUUC	19	4885
  CCR5-2031	+	AGAUCUGGUAAAGAUGAUUC	20	4886
  CCR5-2032	+	GAGAUCUGGUAAAGAUGAUUC	21	4887
  CCR5-2033	+	UGAGAUCUGGUAAAGAUGAUUC	22	4888
  CCR5-2034	+	UUGAGAUCUGGUAAAGAUGAUUC	23	4889
  CCR5-2035	+	UUUGAGAUCUGGUAAAGAUGAUUC	24	4890
  CCR5-2036	+	UUGCCCACAAAACCAAAG	18	4891
  CCR5-2037	+	GUUGCCCACAAAACCAAAG	19	4892
  CCR5-877	+	UGUUGCCCACAAAACCAAAG	20	4893
  CCR5-2038	+	AUGUUGCCCACAAAACCAAAG	21	4894
  CCR5-2039	+	CAUGUUGCCCACAAAACCAAAG	22	4895
  CCR5-2040	+	GCAUGUUGCCCACAAAACCAAAG	23	4896
  CCR5-2041	+	AGCAUGUUGCCCACAAAACCAAAG	24	4897
  CCR5-2042	+	CCAGAAGGGGACAGUAAG	18	4898
  CCR5-2043	+	CCCAGAAGGGGACAGUAAG	19	4899
  CCR5-870	+	GCCCAGAAGGGGACAGUAAG	20	4900
  CCR5-2044	+	AGCCCAGAAGGGGACAGUAAG	21	4901
  CCR5-2045	+	GAGCCCAGAAGGGGACAGUAAG	22	4902
  CCR5-2046	+	UGAGCCCAGAAGGGGACAGUAAG	23	4903
  CCR5-2047	+	GUGAGCCCAGAAGGGGACAGUAAG	24	4904
  CCR5-2048	+	GCAGCAUAGUGAGCCCAG	18	4905
  CCR5-2049	+	GGCAGCAUAGUGAGCCCAG	19	4906
  CCR5-866	+	CGGCAGCAUAGUGAGCCCAG	20	4907
  CCR5-2050	+	GCGGCAGCAUAGUGAGCCCAG	21	4908
  CCR5-2051	+	GGCGGCAGCAUAGUGAGCCCAG	22	4909
  CCR5-2052	+	GGGCGGCAGCAUAGUGAGCCCAG	23	4910
  CCR5-2053	+	UGGGCGGCAGCAUAGUGAGCCCAG	24	4911
  CCR5-2054	+	AUGAAGAAGAUUCCAGAG	18	4912
  CCR5-2055	+	GAUGAAGAAGAUUCCAGAG	19	4913
  CCR5-860	+	UGAUGAAGAAGAUUCCAGAG	20	4914
  CCR5-2056	+	AUGAUGAAGAAGAUUCCAGAG	21	4915
  CCR5-2057	+	GAUGAUGAAGAAGAUUCCAGAG	22	4916
  CCR5-2058	+	GGAUGAUGAAGAAGAUUCCAGAG	23	4917
  CCR5-2059	+	AGGAUGAUGAAGAAGAUUCCAGAG	24	4918
  CCR5-2060	+	GAACACCAGUGAGUAGAG	18	4919
  CCR5-2061	+	UGAACACCAGUGAGUAGAG	19	4920
  CCR5-92	+	AUGAACACCAGUGAGUAGAG	20	4921
  CCR5-2062	+	GAUGAACACCAGUGAGUAGAG	21	4922
  CCR5-2063	+	AGAUGAACACCAGUGAGUAGAG	22	4923
  CCR5-2064	+	AAGAUGAACACCAGUGAGUAGAG	23	4924
  CCR5-2065	+	AAAGAUGAACACCAGUGAGUAGAG	24	4925
  CCR5-2066	+	GUAGAGCGGAGGCAGGAG	18	4926
  CCR5-2067	+	AGUAGAGCGGAGGCAGGAG	19	4927
  CCR5-884	+	GAGUAGAGCGGAGGCAGGAG	20	4928
  CCR5-2068	+	UGAGUAGAGCGGAGGCAGGAG	21	4929
  CCR5-2069	+	GUGAGUAGAGCGGAGGCAGGAG	22	4930
  CCR5-2070	+	AGUGAGUAGAGCGGAGGCAGGAG	23	4931
  CCR5-2071	+	CAGUGAGUAGAGCGGAGGCAGGAG	24	4932
  CCR5-2072	+	AAGAUGAACACCAGUGAG	18	4933
  CCR5-2073	+	AAAGAUGAACACCAGUGAG	19	4934
  CCR5-879	+	CAAAGAUGAACACCAGUGAG	20	4935
  CCR5-2074	+	CCAAAGAUGAACACCAGUGAG	21	4936
  CCR5-2075	+	ACCAAAGAUGAACACCAGUGAG	22	4937
  CCR5-2076	+	AACCAAAGAUGAACACCAGUGAG	23	4938
  CCR5-2077	+	AAACCAAAGAUGAACACCAGUGAG	24	4939
  CCR5-2078	+	AGGUCAGAGAUGGCCAGG	18	4940
  CCR5-2079	+	CAGGUCAGAGAUGGCCAGG	19	4941
  CCR5-873	+	ACAGGUCAGAGAUGGCCAGG	20	4942
  CCR5-2080	+	AACAGGUCAGAGAUGGCCAGG	21	4943
  CCR5-2081	+	AAACAGGUCAGAGAUGGCCAGG	22	4944
  CCR5-2082	+	AAAACAGGUCAGAGAUGGCCAGG	23	4945
  CCR5-2083	+	AAAAACAGGUCAGAGAUGGCCAGG	24	4946
  CCR5-2084	+	CUUUUGCAGUUUAUCAGG	18	4947
  CCR5-2085	+	CCUUUUGCAGUUUAUCAGG	19	4948
  CCR5-875	+	GCCUUUUGCAGUUUAUCAGG	20	4949
  CCR5-2086	+	AGCCUUUUGCAGUUUAUCAGG	21	4950
  CCR5-2087	+	CAGCCUUUUGCAGUUUAUCAGG	22	4951
  CCR5-2088	+	UCAGCCUUUUGCAGUUUAUCAGG	23	4952
  CCR5-2089	+	UUCAGCCUUUUGCAGUUUAUCAGG	24	4953
  CCR5-2090	+	CAGUGAGUAGAGCGGAGG	18	4954
  CCR5-2091	+	CCAGUGAGUAGAGCGGAGG	19	4955
  CCR5-882	+	ACCAGUGAGUAGAGCGGAGG	20	4956
  CCR5-2092	+	CACCAGUGAGUAGAGCGGAGG	21	4957
  CCR5-2093	+	ACACCAGUGAGUAGAGCGGAGG	22	4958
  CCR5-2094	+	AACACCAGUGAGUAGAGCGGAGG	23	4959
  CCR5-2095	+	GAACACCAGUGAGUAGAGCGGAGG	24	4960
  CCR5-2096	+	GGUAAAGAUGAUUCCUGG	18	4961
  CCR5-2097	+	UGGUAAAGAUGAUUCCUGG	19	4962
  CCR5-2098	+	CUGGUAAAGAUGAUUCCUGG	20	4963
  CCR5-2099	+	UCUGGUAAAGAUGAUUCCUGG	21	4964
  CCR5-2100	+	AUCUGGUAAAGAUGAUUCCUGG	22	4965
  CCR5-2101	+	GAUCUGGUAAAGAUGAUUCCUGG	23	4966
  CCR5-2102	+	AGAUCUGGUAAAGAUGAUUCCUGG	24	4967
  CCR5-2103	+	UUCACAUUGAUUUUUUGG	18	4968
  CCR5-2104	+	CUUCACAUUGAUUUUUUGG	19	4969
  CCR5-885	+	GCUUCACAUUGAUUUUUUGG	20	4970
  CCR5-2105	+	UGCUUCACAUUGAUUUUUUGG	21	4971
  CCR5-2106	+	UUGCUUCACAUUGAUUUUUUGG	22	4972
  CCR5-2107	+	UUUGCUUCACAUUGAUUUUUUGG	23	4973
  CCR5-2108	+	AUUUGCUUCACAUUGAUUUUUUGG	24	4974
  CCR5-2109	+	UCGAUUGUCAGGAGGAUG	18	4975
  CCR5-2110	+	AUCGAUUGUCAGGAGGAUG	19	4976
  CCR5-856	+	UAUCGAUUGUCAGGAGGAUG	20	4977
  CCR5-2111	+	CUAUCGAUUGUCAGGAGGAUG	21	4978
  CCR5-2112	+	CCUAUCGAUUGUCAGGAGGAUG	22	4979
  CCR5-2113	+	ACCUAUCGAUUGUCAGGAGGAUG	23	4980
  CCR5-2114	+	UACCUAUCGAUUGUCAGGAGGAUG	24	4981
  CCR5-2115	+	AUUGUCAGGAGGAUGAUG	18	4982
  CCR5-2116	+	GAUUGUCAGGAGGAUGAUG	19	4983
  CCR5-857	+	CGAUUGUCAGGAGGAUGAUG	20	4984
  CCR5-2117	+	UCGAUUGUCAGGAGGAUGAUG	21	4985
  CCR5-2118	+	AUCGAUUGUCAGGAGGAUGAUG	22	4986
  CCR5-2119	+	UAUCGAUUGUCAGGAGGAUGAUG	23	4987
  CCR5-2120	+	CUAUCGAUUGUCAGGAGGAUGAUG	24	4988
  CCR5-2121	+	CUGGUAAAGAUGAUUCCU	18	4989
  CCR5-2122	+	UCUGGUAAAGAUGAUUCCU	19	4990
  CCR5-1877	+	AUCUGGUAAAGAUGAUUCCU	20	4991
  CCR5-2123	+	GAUCUGGUAAAGAUGAUUCCU	21	4992
  CCR5-2124	+	AGAUCUGGUAAAGAUGAUUCCU	22	4993
  CCR5-2125	+	GAGAUCUGGUAAAGAUGAUUCCU	23	4994
  CCR5-2126	+	UGAGAUCUGGUAAAGAUGAUUCCU	24	4995
  CCR5-2127	+	AGCCCAGAAGGGGACAGU	18	4996
  CCR5-2128	+	GAGCCCAGAAGGGGACAGU	19	4997
  CCR5-869	+	UGAGCCCAGAAGGGGACAGU	20	4998
  CCR5-2129	+	GUGAGCCCAGAAGGGGACAGU	21	4999
  CCR5-2130	+	AGUGAGCCCAGAAGGGGACAGU	22	5000
  CCR5-2131	+	UAGUGAGCCCAGAAGGGGACAGU	23	5001
  CCR5-2132	+	AUAGUGAGCCCAGAAGGGGACAGU	24	5002
  CCR5-2133	+	UAAGAAGGAAAAACAGGU	18	5003
  CCR5-2134	+	GUAAGAAGGAAAAACAGGU	19	5004
  CCR5-872	+	AGUAAGAAGGAAAAACAGGU	20	5005
  CCR5-2135	+	CAGUAAGAAGGAAAAACAGGU	21	5006
  CCR5-2136	+	ACAGUAAGAAGGAAAAACAGGU	22	5007
  CCR5-2137	+	GACAGUAAGAAGGAAAAACAGGU	23	5008
  CCR5-2138	+	GGACAGUAAGAAGGAAAAACAGGU	24	5009
  CCR5-2139	+	CAGGUACCUAUCGAUUGU	18	5010
  CCR5-2140	+	CCAGGUACCUAUCGAUUGU	19	5011
  CCR5-853	+	GCCAGGUACCUAUCGAUUGU	20	5012
  CCR5-2141	+	AGCCAGGUACCUAUCGAUUGU	21	5013
  CCR5-2142	+	CAGCCAGGUACCUAUCGAUUGU	22	5014
  CCR5-2143	+	ACAGCCAGGUACCUAUCGAUUGU	23	5015
  CCR5-2144	+	GACAGCCAGGUACCUAUCGAUUGU	24	5016
  CCR5-2145	+	GUAAUGAAGACCUUCUUU	18	5017
  CCR5-2146	+	UGUAAUGAAGACCUUCUUU	19	5018
  CCR5-1657	+	GUGUAAUGAAGACCUUCUUU	20	5019
  CCR5-2147	−	UCUUUACCAGAUCUCAAA	18	5020
  CCR5-2148	−	AUCUUUACCAGAUCUCAAA	19	5021
  CCR5-2149	−	CAUCUUUACCAGAUCUCAAA	20	5022
  CCR5-2150	−	UCAUCUUUACCAGAUCUCAAA	21	5023
  CCR5-2151	−	AUCAUCUUUACCAGAUCUCAAA	22	5024
  CCR5-2152	−	AAUCAUCUUUACCAGAUCUCAAA	23	5025
  CCR5-2153	−	GAAUCAUCUUUACCAGAUCUCAAA	24	5026
  CCR5-2154	−	GACAUCAAUUAUUAUACA	18	5027
  CCR5-2155	−	UGACAUCAAUUAUUAUACA	19	5028
  CCR5-812	−	AUGACAUCAAUUAUUAUACA	20	5029
  CCR5-2156	−	UAUGACAUCAAUUAUUAUACA	21	5030
  CCR5-2157	−	CUAUGACAUCAAUUAUUAUACA	22	5031
  CCR5-2158	−	UCUAUGACAUCAAUUAUUAUACA	23	5032
  CCR5-2159	−	AUCUAUGACAUCAAUUAUUAUACA	24	5033
  CCR5-2160	−	UCACUAUGCUGCCGCCCA	18	5034
  CCR5-2161	−	CUCACUAUGCUGCCGCCCA	19	5035
  CCR5-819	−	GCUCACUAUGCUGCCGCCCA	20	5036
  CCR5-2162	−	GGCUCACUAUGCUGCCGCCCA	21	5037
  CCR5-2163	−	GGGCUCACUAUGCUGCCGCCCA	22	5038
  CCR5-2164	−	UGGGCUCACUAUGCUGCCGCCCA	23	5039
  CCR5-2165	−	CUGGGCUCACUAUGCUGCCGCCCA	24	5040
  CCR5-2166	−	CAAUGUGUCAACUCUUGA	18	5041
  CCR5-2167	−	ACAAUGUGUCAACUCUUGA	19	5042
  CCR5-823	−	UACAAUGUGUCAACUCUUGA	20	5043
  CCR5-2168	−	AUACAAUGUGUCAACUCUUGA	21	5044
  CCR5-2169	−	AAUACAAUGUGUCAACUCUUGA	22	5045
  CCR5-2170	−	AAAUACAAUGUGUCAACUCUUGA	23	5046
  CCR5-2171	−	GAAAUACAAUGUGUCAACUCUUGA	24	5047
  CCR5-2172	−	CUGUGUUUGCGUCUCUCC	18	5048
  CCR5-2173	−	GCUGUGUUUGCGUCUCUCC	19	5049
  CCR5-830	−	GGCUGUGUUUGCGUCUCUCC	20	5050
  CCR5-2174	−	UGGCUGUGUUUGCGUCUCUCC	21	5051
  CCR5-2175	−	GUGGCUGUGUUUGCGUCUCUCC	22	5052
  CCR5-2176	−	GGUGGCUGUGUUUGCGUCUCUCC	23	5053
  CCR5-2177	−	UGGUGGCUGUGUUUGCGUCUCUCC	24	5054
  CCR5-2178	−	UGUGUUUGCUUUAAAAGC	18	5055
  CCR5-2179	−	CUGUGUUUGCUUUAAAAGC	19	5056
  CCR5-826	−	GCUGUGUUUGCUUUAAAAGC	20	5057
  CCR5-2180	−	UGCUGUGUUUGCUUUAAAAGC	21	5058
  CCR5-2181	−	AUGCUGUGUUUGCUUUAAAAGC	22	5059
  CCR5-2182	−	CAUGCUGUGUUUGCUUUAAAAGC	23	5060
  CCR5-2183	−	CCAUGCUGUGUUUGCUUUAAAAGC	24	5061
  CCR5-2184	−	CACUAUGCUGCCGCCCAG	18	5062
  CCR5-2185	−	UCACUAUGCUGCCGCCCAG	19	5063
  CCR5-74	−	CUCACUAUGCUGCCGCCCAG	20	5064
  CCR5-2186	−	GCUCACUAUGCUGCCGCCCAG	21	5065
  CCR5-2187	−	GGCUCACUAUGCUGCCGCCCAG	22	5066
  CCR5-2188	−	GGGCUCACUAUGCUGCCGCCCAG	23	5067
  CCR5-2189	−	UGGGCUCACUAUGCUGCCGCCCAG	24	5068
  CCR5-2190	−	CUGAUAAACUGCAAAAGG	18	5069
  CCR5-2191	−	CCUGAUAAACUGCAAAAGG	19	5070
  CCR5-816	−	UCCUGAUAAACUGCAAAAGG	20	5071
  CCR5-2192	−	AUCCUGAUAAACUGCAAAAGG	21	5072
  CCR5-2193	−	CAUCCUGAUAAACUGCAAAAGG	22	5073
  CCR5-2194	−	UCAUCCUGAUAAACUGCAAAAGG	23	5074
  CCR5-2195	−	CUCAUCCUGAUAAACUGCAAAAGG	24	5075
  CCR5-2196	−	UUUUUAUUUAUGCACAGG	18	5076
  CCR5-2197	−	CUUUUUAUUUAUGCACAGG	19	5077
  CCR5-2198	−	ACUUUUUAUUUAUGCACAGG	20	5078
  CCR5-2199	−	UUUUAUUUAUGCACAGGG	18	5079
  CCR5-2200	−	UUUUUAUUUAUGCACAGGG	19	5080
  CCR5-1876	−	CUUUUUAUUUAUGCACAGGG	20	5081
  CCR5-2201	−	AUAAACUGCAAAAGGCUG	18	5082
  CCR5-2202	−	GAUAAACUGCAAAAGGCUG	19	5083
  CCR5-817	−	UGAUAAACUGCAAAAGGCUG	20	5084
  CCR5-2203	−	CUGAUAAACUGCAAAAGGCUG	21	5085
  CCR5-2204	−	CCUGAUAAACUGCAAAAGGCUG	22	5086
  CCR5-2205	−	UCCUGAUAAACUGCAAAAGGCUG	23	5087
  CCR5-2206	−	AUCCUGAUAAACUGCAAAAGGCUG	24	5088
  CCR5-2207	−	CCCUGCCAAAAAAUCAAU	18	5089
  CCR5-2208	−	GCCCUGCCAAAAAAUCAAU	19	5090
  CCR5-814	−	AGCCCUGCCAAAAAAUCAAU	20	5091
  CCR5-2209	−	GAGCCCUGCCAAAAAAUCAAU	21	5092
  CCR5-2210	−	GGAGCCCUGCCAAAAAAUCAAU	22	5093
  CCR5-2211	−	CGGAGCCCUGCCAAAAAAUCAAU	23	5094
  CCR5-2212	−	UCGGAGCCCUGCCAAAAAAUCAAU	24	5095
  CCR5-2213	−	ACAUCAAUUAUUAUACAU	18	5096
  CCR5-2214	−	GACAUCAAUUAUUAUACAU	19	5097
  CCR5-67	−	UGACAUCAAUUAUUAUACAU	20	5098
  CCR5-2215	−	AUGACAUCAAUUAUUAUACAU	21	5099
  CCR5-2216	−	UAUGACAUCAAUUAUUAUACAU	22	5100
  CCR5-2217	−	CUAUGACAUCAAUUAUUAUACAU	23	5101
  CCR5-2218	−	UCUAUGACAUCAAUUAUUAUACAU	24	5102
  CCR5-2219	−	CUGCCGCCCAGUGGGACU	18	5103
  CCR5-2220	−	GCUGCCGCCCAGUGGGACU	19	5104
  CCR5-821	−	UGCUGCCGCCCAGUGGGACU	20	5105
  CCR5-2221	−	AUGCUGCCGCCCAGUGGGACU	21	5106
  CCR5-2222	−	UAUGCUGCCGCCCAGUGGGACU	22	5107
  CCR5-2223	−	CUAUGCUGCCGCCCAGUGGGACU	23	5108
  CCR5-2224	−	ACUAUGCUGCCGCCCAGUGGGACU	24	5109
  CCR5-2225	−	AAGCCAGGACGGUCACCU	18	5110
  CCR5-2226	−	AAAGCCAGGACGGUCACCU	19	5111
  CCR5-827	−	AAAAGCCAGGACGGUCACCU	20	5112
  CCR5-2227	−	UAAAAGCCAGGACGGUCACCU	21	5113
  CCR5-2228	−	UUAAAAGCCAGGACGGUCACCU	22	5114
  CCR5-2229	−	UUUAAAAGCCAGGACGGUCACCU	23	5115
  CCR5-2230	−	CUUUAAAAGCCAGGACGGUCACCU	24	5116
  CCR5-2231	−	AUUUUAUAGGCUUCUUCU	18	5117
  CCR5-2232	−	UAUUUUAUAGGCUUCUUCU	19	5118
  CCR5-824	−	CUAUUUUAUAGGCUUCUUCU	20	5119
  CCR5-2233	−	UCUAUUUUAUAGGCUUCUUCU	21	5120
  CCR5-2234	−	CUCUAUUUUAUAGGCUUCUUCU	22	5121
  CCR5-2235	−	GCUCUAUUUUAUAGGCUUCUUCU	23	5122
  CCR5-2236	−	GGCUCUAUUUUAUAGGCUUCUUCU	24	5123
  CCR5-2237	−	UGCCGCCCAGUGGGACUU	18	5124
  CCR5-2238	−	CUGCCGCCCAGUGGGACUU	19	5125
  CCR5-43	−	GCUGCCGCCCAGUGGGACUU	20	5126
  CCR5-2239	−	UGCUGCCGCCCAGUGGGACUU	21	5127
  CCR5-2240	−	AUGCUGCCGCCCAGUGGGACUU	22	5128
  CCR5-2241	−	UAUGCUGCCGCCCAGUGGGACUU	23	5129
  CCR5-2242	−	CUAUGCUGCCGCCCAGUGGGACUU	24	5130
  CCR5-2243	−	CCUUCUUACUGUCCCCUU	18	5131
  CCR5-2244	−	UCCUUCUUACUGUCCCCUU	19	5132
  CCR5-818	−	UUCCUUCUUACUGUCCCCUU	20	5133
  CCR5-2245	−	UUUCCUUCUUACUGUCCCCUU	21	5134
  CCR5-2246	−	UUUUCCUUCUUACUGUCCCCUU	22	5135
  CCR5-2247	−	UUUUUCCUUCUUACUGUCCCCUU	23	5136
  CCR5-2248	−	GUUUUUCCUUCUUACUGUCCCCUU	24	5137
  CCR5-2249	−	GUGUUCAUCUUUGGUUUU	18	5138
  CCR5-2250	−	GGUGUUCAUCUUUGGUUUU	19	5139
  CCR5-815	−	UGGUGUUCAUCUUUGGUUUU	20	5140
  CCR5-2251	−	CUGGUGUUCAUCUUUGGUUUU	21	5141
  CCR5-2252	−	ACUGGUGUUCAUCUUUGGUUUU	22	5142
  CCR5-2253	−	CACUGGUGUUCAUCUUUGGUUUU	23	5143
  CCR5-2254	−	UCACUGGUGUUCAUCUUUGGUUUU	24	5144

• Table 3E provides exemplary targeting domains for knocking out the CCR5 gene selected according to the fifth tier parameters. The targeting domains fall in the coding sequence of the gene, downstream of the first 500 bp of coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon of the gene and PAM is NNGRRV. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 3E
  5th Tier

  gRNA	DNA		Target Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO

  CCR5-2339	+	GAGCCUCUUGCUGGAAAA	18	5145
  CCR5-2340	+	GGAGCCUCUUGCUGGAAAA	19	5146
  CCR5-1619	+	GGGAGCCUCUUGCUGGAAAA	20	5147
  CCR5-2341	+	CGGGAGCCUCUUGCUGGAAAA	21	5148
  CCR5-2342	+	UCGGGAGCCUCUUGCUGGAAAA	22	5149
  CCR5-2343	+	CUCGGGAGCCUCUUGCUGGAAAA	23	5150
  CCR5-2344	+	GCUCGGGAGCCUCUUGCUGGAAAA	24	5151
  CCR5-2345	+	AUACUGACUGUAUGGAAA	18	5152
  CCR5-2346	+	GAUACUGACUGUAUGGAAA	19	5153
  CCR5-1654	+	UGAUACUGACUGUAUGGAAA	20	5154
  CCR5-2347	+	UUGAUACUGACUGUAUGGAAA	21	5155
  CCR5-2348	+	AUUGAUACUGACUGUAUGGAAA	22	5156
  CCR5-2349	+	AAUUGAUACUGACUGUAUGGAAA	23	5157
  CCR5-2350	+	GAAUUGAUACUGACUGUAUGGAAA	24	5158
  CCR5-2351	+	CAGUGGAUCGGGUGUAAA	18	5159
  CCR5-2352	+	CCAGUGGAUCGGGUGUAAA	19	5160
  CCR5-1613	+	CCCAGUGGAUCGGGUGUAAA	20	5161
  CCR5-2353	+	CCCCAGUGGAUCGGGUGUAAA	21	5162
  CCR5-2354	+	UCCCCAGUGGAUCGGGUGUAAA	22	5163
  CCR5-2355	+	CUCCCCAGUGGAUCGGGUGUAAA	23	5164
  CCR5-2356	+	GCUCCCCAGUGGAUCGGGUGUAAA	24	5165
  CCR5-2357	+	GUUGUAGGGAGCCCAGAA	18	5166
  CCR5-2358	+	UGUUGUAGGGAGCCCAGAA	19	5167
  CCR5-1642	+	AUGUUGUAGGGAGCCCAGAA	20	5168
  CCR5-2359	+	AAUGUUGUAGGGAGCCCAGAA	21	5169
  CCR5-2360	+	CAAUGUUGUAGGGAGCCCAGAA	22	5170
  CCR5-2361	+	ACAAUGUUGUAGGGAGCCCAGAA	23	5171
  CCR5-2362	+	GACAAUGUUGUAGGGAGCCCAGAA	24	5172
  CCR5-2363	+	CUUCUUCUCAUUUCGACA	18	5173
  CCR5-2364	+	UCUUCUUCUCAUUUCGACA	19	5174
  CCR5-1644	+	CUCUUCUUCUCAUUUCGACA	20	5175
  CCR5-2365	+	CCUCUUCUUCUCAUUUCGACA	21	5176
  CCR5-2366	+	GCCUCUUCUUCUCAUUUCGACA	22	5177
  CCR5-2367	+	UGCCUCUUCUUCUCAUUUCGACA	23	5178
  CCR5-2368	+	GUGCCUCUUCUUCUCAUUUCGACA	24	5179
  CCR5-2369	+	GAAUUGAUACUGACUGUA	18	5180
  CCR5-2370	+	AGAAUUGAUACUGACUGUA	19	5181
  CCR5-699	+	CAGAAUUGAUACUGACUGUA	20	5182
  CCR5-2371	+	CCAGAAUUGAUACUGACUGUA	21	5183
  CCR5-2372	+	UCCAGAAUUGAUACUGACUGUA	22	5184
  CCR5-2373	+	UUCCAGAAUUGAUACUGACUGUA	23	5185
  CCR5-2374	+	CUUCCAGAAUUGAUACUGACUGUA	24	5186
  CCR5-2375	+	AUGAGAGCUGCAGGUGUA	18	5187
  CCR5-2376	+	AAUGAGAGCUGCAGGUGUA	19	5188
  CCR5-1656	+	AAAUGAGAGCUGCAGGUGUA	20	5189
  CCR5-2377	+	AAAAUGAGAGCUGCAGGUGUA	21	5190
  CCR5-2378	+	GAAAAUGAGAGCUGCAGGUGUA	22	5191
  CCR5-2379	+	GGAAAAUGAGAGCUGCAGGUGUA	23	5192
  CCR5-2380	+	UGGAAAAUGAGAGCUGCAGGUGUA	24	5193
  CCR5-2381	+	GAGAAGGACAAUGUUGUA	18	5194
  CCR5-2382	+	GGAGAAGGACAAUGUUGUA	19	5195
  CCR5-693	+	AGGAGAAGGACAAUGUUGUA	20	5196
  CCR5-2383	+	CAGGAGAAGGACAAUGUUGUA	21	5197
  CCR5-2384	+	UCAGGAGAAGGACAAUGUUGUA	22	5198
  CCR5-2385	+	UUCAGGAGAAGGACAAUGUUGUA	23	5199
  CCR5-2386	+	GUUCAGGAGAAGGACAAUGUUGUA	24	5200
  CCR5-2387	+	ACAAUGUUGUAGGGAGCC	18	5201
  CCR5-2388	+	GACAAUGUUGUAGGGAGCC	19	5202
  CCR5-1640	+	GGACAAUGUUGUAGGGAGCC	20	5203
  CCR5-2389	+	AGGACAAUGUUGUAGGGAGCC	21	5204
  CCR5-2390	+	AAGGACAAUGUUGUAGGGAGCC	22	5205
  CCR5-2391	+	GAAGGACAAUGUUGUAGGGAGCC	23	5206
  CCR5-2392	+	AGAAGGACAAUGUUGUAGGGAGCC	24	5207
  CCR5-2393	+	UUCAGGCCAAAGAAUUCC	18	5208
  CCR5-2394	+	AUUCAGGCCAAAGAAUUCC	19	5209
  CCR5-688	+	UAUUCAGGCCAAAGAAUUCC	20	5210
  CCR5-2395	+	UUAUUCAGGCCAAAGAAUUCC	21	5211
  CCR5-2396	+	AUUAUUCAGGCCAAAGAAUUCC	22	5212
  CCR5-2397	+	AAUUAUUCAGGCCAAAGAAUUCC	23	5213
  CCR5-2398	+	CAAUUAUUCAGGCCAAAGAAUUCC	24	5214
  CCR5-1999	+	UCUGGUAAAGAUGAUUCC	18	5215
  CCR5-2000	+	AUCUGGUAAAGAUGAUUCC	19	5216
  CCR5-1874	+	GAUCUGGUAAAGAUGAUUCC	20	5217
  CCR5-2001	+	AGAUCUGGUAAAGAUGAUUCC	21	5218
  CCR5-2002	+	GAGAUCUGGUAAAGAUGAUUCC	22	5219
  CCR5-2003	+	UGAGAUCUGGUAAAGAUGAUUCC	23	5220
  CCR5-2004	+	UUGAGAUCUGGUAAAGAUGAUUCC	24	5221
  CCR5-2399	+	UAAACUGAGCUUGCUCGC	18	5222
  CCR5-2400	+	GUAAACUGAGCUUGCUCGC	19	5223
  CCR5-1614	+	UGUAAACUGAGCUUGCUCGC	20	5224
  CCR5-2401	+	GUGUAAACUGAGCUUGCUCGC	21	5225
  CCR5-2402	+	GGUGUAAACUGAGCUUGCUCGC	22	5226
  CCR5-2403	+	GGGUGUAAACUGAGCUUGCUCGC	23	5227
  CCR5-2404	+	CGGGUGUAAACUGAGCUUGCUCGC	24	5228
  CCR5-2405	+	CGCUCGGGAGCCUCUUGC	18	5229
  CCR5-2406	+	UCGCUCGGGAGCCUCUUGC	19	5230
  CCR5-678	+	CUCGCUCGGGAGCCUCUUGC	20	5231
  CCR5-2407	+	GCUCGCUCGGGAGCCUCUUGC	21	5232
  CCR5-2408	+	UGCUCGCUCGGGAGCCUCUUGC	22	5233
  CCR5-2409	+	UUGCUCGCUCGGGAGCCUCUUGC	23	5234
  CCR5-2410	+	CUUGCUCGCUCGGGAGCCUCUUGC	24	5235
  CCR5-2411	+	AACUGAGCUUGCUCGCUC	18	5236
  CCR5-2412	+	AAACUGAGCUUGCUCGCUC	19	5237
  CCR5-677	+	UAAACUGAGCUUGCUCGCUC	20	5238
  CCR5-2413	+	GUAAACUGAGCUUGCUCGCUC	21	5239
  CCR5-2414	+	UGUAAACUGAGCUUGCUCGCUC	22	5240
  CCR5-2415	+	GUGUAAACUGAGCUUGCUCGCUC	23	5241
  CCR5-2416	+	GGUGUAAACUGAGCUUGCUCGCUC	24	5242
  CCR5-2417	+	AUGACUAUCUUUAAUGUC	18	5243
  CCR5-2418	+	GAUGACUAUCUUUAAUGUC	19	5244
  CCR5-698	+	AGAUGACUAUCUUUAAUGUC	20	5245
  CCR5-2419	+	AAGAUGACUAUCUUUAAUGUC	21	5246
  CCR5-2420	+	CAAGAUGACUAUCUUUAAUGUC	22	5247
  CCR5-2421	+	CCAAGAUGACUAUCUUUAAUGUC	23	5248
  CCR5-2422	+	CCCAAGAUGACUAUCUUUAAUGUC	24	5249
  CCR5-2423	+	AUUCAGGCCAAAGAAUUC	18	5250
  CCR5-2424	+	UAUUCAGGCCAAAGAAUUC	19	5251
  CCR5-1631	+	UUAUUCAGGCCAAAGAAUUC	20	5252
  CCR5-2425	+	AUUAUUCAGGCCAAAGAAUUC	21	5253
  CCR5-2426	+	AAUUAUUCAGGCCAAAGAAUUC	22	5254
  CCR5-2427	+	CAAUUAUUCAGGCCAAAGAAUUC	23	5255
  CCR5-2428	+	GCAAUUAUUCAGGCCAAAGAAUUC	24	5256
  CCR5-2029	+	AUCUGGUAAAGAUGAUUC	18	5257
  CCR5-2030	+	GAUCUGGUAAAGAUGAUUC	19	5258
  CCR5-2031	+	AGAUCUGGUAAAGAUGAUUC	20	5259
  CCR5-2032	+	GAGAUCUGGUAAAGAUGAUUC	21	5260
  CCR5-2033	+	UGAGAUCUGGUAAAGAUGAUUC	22	5261
  CCR5-2034	+	UUGAGAUCUGGUAAAGAUGAUUC	23	5262
  CCR5-2035	+	UUUGAGAUCUGGUAAAGAUGAUUC	24	5263
  CCR5-2429	+	AAUUCCUGGAAGGUGUUC	18	5264
  CCR5-2430	+	GAAUUCCUGGAAGGUGUUC	19	5265
  CCR5-690	+	AGAAUUCCUGGAAGGUGUUC	20	5266
  CCR5-2431	+	AAGAAUUCCUGGAAGGUGUUC	21	5267
  CCR5-2432	+	AAAGAAUUCCUGGAAGGUGUUC	22	5268
  CCR5-2433	+	CAAAGAAUUCCUGGAAGGUGUUC	23	5269
  CCR5-2434	+	CCAAAGAAUUCCUGGAAGGUGUUC	24	5270
  CCR5-2435	+	AUGUUGUAGGGAGCCCAG	18	5271
  CCR5-2436	+	AAUGUUGUAGGGAGCCCAG	19	5272
  CCR5-1641	+	CAAUGUUGUAGGGAGCCCAG	20	5273
  CCR5-2437	+	ACAAUGUUGUAGGGAGCCCAG	21	5274
  CCR5-2438	+	GACAAUGUUGUAGGGAGCCCAG	22	5275
  CCR5-2439	+	GGACAAUGUUGUAGGGAGCCCAG	23	5276
  CCR5-2440	+	AGGACAAUGUUGUAGGGAGCCCAG	24	5277
  CCR5-2441	+	UUCCUGGAAGGUGUUCAG	18	5278
  CCR5-2442	+	AUUCCUGGAAGGUGUUCAG	19	5279
  CCR5-1635	+	AAUUCCUGGAAGGUGUUCAG	20	5280
  CCR5-2443	+	GAAUUCCUGGAAGGUGUUCAG	21	5281
  CCR5-2444	+	AGAAUUCCUGGAAGGUGUUCAG	22	5282
  CCR5-2445	+	AAGAAUUCCUGGAAGGUGUUCAG	23	5283
  CCR5-2446	+	AAAGAAUUCCUGGAAGGUGUUCAG	24	5284
  CCR5-2447	+	CUGGAAGGUGUUCAGGAG	18	5285
  CCR5-2448	+	CCUGGAAGGUGUUCAGGAG	19	5286
  CCR5-1636	+	UCCUGGAAGGUGUUCAGGAG	20	5287
  CCR5-2449	+	UUCCUGGAAGGUGUUCAGGAG	21	5288
  CCR5-2450	+	AUUCCUGGAAGGUGUUCAGGAG	22	5289
  CCR5-2451	+	AAUUCCUGGAAGGUGUUCAGGAG	23	5290
  CCR5-2452	+	GAAUUCCUGGAAGGUGUUCAGGAG	24	5291
  CCR5-2453	+	GACCAUGACAAGCAGCGG	18	5292
  CCR5-2454	+	UGACCAUGACAAGCAGCGG	19	5293
  CCR5-1648	+	AUGACCAUGACAAGCAGCGG	20	5294
  CCR5-2455	+	GAUGACCAUGACAAGCAGCGG	21	5295
  CCR5-2456	+	AGAUGACCAUGACAAGCAGCGG	22	5296
  CCR5-2457	+	CAGAUGACCAUGACAAGCAGCGG	23	5297
  CCR5-2458	+	GCAGAUGACCAUGACAAGCAGCGG	24	5298
  CCR5-2096	+	GGUAAAGAUGAUUCCUGG	18	5299
  CCR5-2097	+	UGGUAAAGAUGAUUCCUGG	19	5300
  CCR5-2098	+	CUGGUAAAGAUGAUUCCUGG	20	5301
  CCR5-2099	+	UCUGGUAAAGAUGAUUCCUGG	21	5302
  CCR5-2100	+	AUCUGGUAAAGAUGAUUCCUGG	22	5303
  CCR5-2101	+	GAUCUGGUAAAGAUGAUUCCUGG	23	5304
  CCR5-2102	+	AGAUCUGGUAAAGAUGAUUCCUGG	24	5305
  CCR5-2459	+	UUGGCAAUGUGCUUUUGG	18	5306
  CCR5-2460	+	UUUGGCAAUGUGCUUUUGG	19	5307
  CCR5-1623	+	GUUUGGCAAUGUGCUUUUGG	20	5308
  CCR5-2461	+	CGUUUGGCAAUGUGCUUUUGG	21	5309
  CCR5-2462	+	GCGUUUGGCAAUGUGCUUUUGG	22	5310
  CCR5-2463	+	AGCGUUUGGCAAUGUGCUUUUGG	23	5311
  CCR5-2464	+	AAGCGUUUGGCAAUGUGCUUUUGG	24	5312
  CCR5-2465	+	CCGACAAAGGCAUAGAUG	18	5313
  CCR5-2466	+	CCCGACAAAGGCAUAGAUG	19	5314
  CCR5-1626	+	CCCCGACAAAGGCAUAGAUG	20	5315
  CCR5-2467	+	UCCCCGACAAAGGCAUAGAUG	21	5316
  CCR5-2468	+	CUCCCCGACAAAGGCAUAGAUG	22	5317
  CCR5-2469	+	UCUCCCCGACAAAGGCAUAGAUG	23	5318
  CCR5-2470	+	UUCUCCCCGACAAAGGCAUAGAUG	24	5319
  CCR5-2471	+	AAAUAAACAAUCAUGAUG	18	5320
  CCR5-2472	+	AAAAUAAACAAUCAUGAUG	19	5321
  CCR5-1643	+	GAAAAUAAACAAUCAUGAUG	20	5322
  CCR5-2473	+	AGAAAAUAAACAAUCAUGAUG	21	5323
  CCR5-2474	+	GAGAAAAUAAACAAUCAUGAUG	22	5324
  CCR5-2475	+	AGAGAAAAUAAACAAUCAUGAUG	23	5325
  CCR5-2476	+	AAGAGAAAAUAAACAAUCAUGAUG	24	5326
  CCR5-2477	+	UCGCUCGGGAGCCUCUUG	18	5327
  CCR5-2478	+	CUCGCUCGGGAGCCUCUUG	19	5328
  CCR5-1617	+	GCUCGCUCGGGAGCCUCUUG	20	5329
  CCR5-2479	+	UGCUCGCUCGGGAGCCUCUUG	21	5330
  CCR5-2480	+	UUGCUCGCUCGGGAGCCUCUUG	22	5331
  CCR5-2481	+	CUUGCUCGCUCGGGAGCCUCUUG	23	5332
  CCR5-2482	+	GCUUGCUCGCUCGGGAGCCUCUUG	24	5333
  CCR5-2483	+	AGGAGAAGGACAAUGUUG	18	5334
  CCR5-2484	+	CAGGAGAAGGACAAUGUUG	19	5335
  CCR5-1637	+	UCAGGAGAAGGACAAUGUUG	20	5336
  CCR5-2485	+	UUCAGGAGAAGGACAAUGUUG	21	5337
  CCR5-2486	+	GUUCAGGAGAAGGACAAUGUUG	22	5338
  CCR5-2487	+	UGUUCAGGAGAAGGACAAUGUUG	23	5339
  CCR5-2488	+	GUGUUCAGGAGAAGGACAAUGUUG	24	5340
  CCR5-2489	+	AAAAUAGAACAGCAUUUG	18	5341
  CCR5-2490	+	GAAAAUAGAACAGCAUUUG	19	5342
  CCR5-1620	+	GGAAAAUAGAACAGCAUUUG	20	5343
  CCR5-2491	+	UGGAAAAUAGAACAGCAUUUG	21	5344
  CCR5-2492	+	CUGGAAAAUAGAACAGCAUUUG	22	5345
  CCR5-2493	+	GCUGGAAAAUAGAACAGCAUUUG	23	5346
  CCR5-2494	+	UGCUGGAAAAUAGAACAGCAUUUG	24	5347
  CCR5-2495	+	ACUGACUGUAUGGAAAAU	18	5348
  CCR5-2496	+	UACUGACUGUAUGGAAAAU	19	5349
  CCR5-1655	+	AUACUGACUGUAUGGAAAAU	20	5350
  CCR5-2497	+	GAUACUGACUGUAUGGAAAAU	21	5351
  CCR5-2498	+	UGAUACUGACUGUAUGGAAAAU	22	5352
  CCR5-2499	+	UUGAUACUGACUGUAUGGAAAAU	23	5353
  CCR5-2500	+	AUUGAUACUGACUGUAUGGAAAAU	24	5354
  CCR5-2501	+	UGCUUUUGGAAGAAGACU	18	5355
  CCR5-2502	+	GUGCUUUUGGAAGAAGACU	19	5356
  CCR5-1624	+	UGUGCUUUUGGAAGAAGACU	20	5357
  CCR5-2503	+	AUGUGCUUUUGGAAGAAGACU	21	5358
  CCR5-2504	+	AAUGUGCUUUUGGAAGAAGACU	22	5359
  CCR5-2505	+	CAAUGUGCUUUUGGAAGAAGACU	23	5360
  CCR5-2506	+	GCAAUGUGCUUUUGGAAGAAGACU	24	5361
  CCR5-2121	+	CUGGUAAAGAUGAUUCCU	18	5362
  CCR5-2122	+	UCUGGUAAAGAUGAUUCCU	19	5363
  CCR5-1877	+	AUCUGGUAAAGAUGAUUCCU	20	5364
  CCR5-2123	+	GAUCUGGUAAAGAUGAUUCCU	21	5365
  CCR5-2124	+	AGAUCUGGUAAAGAUGAUUCCU	22	5366
  CCR5-2125	+	GAGAUCUGGUAAAGAUGAUUCCU	23	5367
  CCR5-2126	+	UGAGAUCUGGUAAAGAUGAUUCCU	24	5368
  CCR5-2507	+	AAACUGAGCUUGCUCGCU	18	5369
  CCR5-2508	+	UAAACUGAGCUUGCUCGCU	19	5370
  CCR5-676	+	GUAAACUGAGCUUGCUCGCU	20	5371
  CCR5-2509	+	UGUAAACUGAGCUUGCUCGCU	21	5372
  CCR5-2510	+	GUGUAAACUGAGCUUGCUCGCU	22	5373
  CCR5-2511	+	GGUGUAAACUGAGCUUGCUCGCU	23	5374
  CCR5-2512	+	GGGUGUAAACUGAGCUUGCUCGCU	24	5375
  CCR5-2513	+	GAUGACUAUCUUUAAUGU	18	5376
  CCR5-2514	+	AGAUGACUAUCUUUAAUGU	19	5377
  CCR5-1649	+	AAGAUGACUAUCUUUAAUGU	20	5378
  CCR5-2515	+	CAAGAUGACUAUCUUUAAUGU	21	5379
  CCR5-2516	+	CCAAGAUGACUAUCUUUAAUGU	22	5380
  CCR5-2517	+	CCCAAGAUGACUAUCUUUAAUGU	23	5381
  CCR5-2518	+	CCCCAAGAUGACUAUCUUUAAUGU	24	5382
  CCR5-2519	+	AGAAUUGAUACUGACUGU	18	5383
  CCR5-2520	+	CAGAAUUGAUACUGACUGU	19	5384
  CCR5-1652	+	CCAGAAUUGAUACUGACUGU	20	5385
  CCR5-2521	+	UCCAGAAUUGAUACUGACUGU	21	5386
  CCR5-2522	+	UUCCAGAAUUGAUACUGACUGU	22	5387
  CCR5-2523	+	CUUCCAGAAUUGAUACUGACUGU	23	5388
  CCR5-2524	+	UCUUCCAGAAUUGAUACUGACUGU	24	5389
  CCR5-2525	+	UAGCUUGGUCCAACCUGU	18	5390
  CCR5-2526	+	AUAGCUUGGUCCAACCUGU	19	5391
  CCR5-1629	+	CAUAGCUUGGUCCAACCUGU	20	5392
  CCR5-2527	+	GCAUAGCUUGGUCCAACCUGU	21	5393
  CCR5-2528	+	UGCAUAGCUUGGUCCAACCUGU	22	5394
  CCR5-2529	+	CUGCAUAGCUUGGUCCAACCUGU	23	5395
  CCR5-2530	+	CCUGCAUAGCUUGGUCCAACCUGU	24	5396
  CCR5-2531	+	GGAGAAGGACAAUGUUGU	18	5397
  CCR5-2532	+	AGGAGAAGGACAAUGUUGU	19	5398
  CCR5-692	+	CAGGAGAAGGACAAUGUUGU	20	5399
  CCR5-2533	+	UCAGGAGAAGGACAAUGUUGU	21	5400
  CCR5-2534	+	UUCAGGAGAAGGACAAUGUUGU	22	5401
  CCR5-2535	+	GUUCAGGAGAAGGACAAUGUUGU	23	5402
  CCR5-2536	+	UGUUCAGGAGAAGGACAAUGUUGU	24	5403
  CCR5-2537	+	GCGUUUGGCAAUGUGCUU	18	5404
  CCR5-2538	+	AGCGUUUGGCAAUGUGCUU	19	5405
  CCR5-1621	+	AAGCGUUUGGCAAUGUGCUU	20	5406
  CCR5-2539	+	GAAGCGUUUGGCAAUGUGCUU	21	5407
  CCR5-2540	+	AGAAGCGUUUGGCAAUGUGCUU	22	5408
  CCR5-2541	+	CAGAAGCGUUUGGCAAUGUGCUU	23	5409
  CCR5-2542	+	GCAGAAGCGUUUGGCAAUGUGCUU	24	5410
  CCR5-2543	+	GAAUUCCUGGAAGGUGUU	18	5411
  CCR5-2544	+	AGAAUUCCUGGAAGGUGUU	19	5412
  CCR5-1633	+	AAGAAUUCCUGGAAGGUGUU	20	5413
  CCR5-2545	+	AAAGAAUUCCUGGAAGGUGUU	21	5414
  CCR5-2546	+	CAAAGAAUUCCUGGAAGGUGUU	22	5415
  CCR5-2547	+	CCAAAGAAUUCCUGGAAGGUGUU	23	5416
  CCR5-2548	+	GCCAAAGAAUUCCUGGAAGGUGUU	24	5417
  CCR5-2549	+	CGUUUGGCAAUGUGCUUU	18	5418
  CCR5-2550	+	GCGUUUGGCAAUGUGCUUU	19	5419
  CCR5-680	+	AGCGUUUGGCAAUGUGCUUU	20	5420
  CCR5-2551	+	AAGCGUUUGGCAAUGUGCUUU	21	5421
  CCR5-2552	+	GAAGCGUUUGGCAAUGUGCUUU	22	5422
  CCR5-2553	+	AGAAGCGUUUGGCAAUGUGCUUU	23	5423
  CCR5-2554	+	CAGAAGCGUUUGGCAAUGUGCUUU	24	5424
  CCR5-2145	+	GUAAUGAAGACCUUCUUU	18	5425
  CCR5-2146	+	UGUAAUGAAGACCUUCUUU	19	5426
  CCR5-1657	+	GUGUAAUGAAGACCUUCUUU	20	5427
  CCR5-2555	+	GGUGUAAUGAAGACCUUCUUU	21	5428
  CCR5-2556	+	AGGUGUAAUGAAGACCUUCUUU	22	5429
  CCR5-2557	+	CAGGUGUAAUGAAGACCUUCUUU	23	5430
  CCR5-2558	+	GCAGGUGUAAUGAAGACCUUCUUU	24	5431
  CCR5-2559	+	AAGACUAAGAGGUAGUUU	18	5432
  CCR5-2560	+	GAAGACUAAGAGGUAGUUU	19	5433
  CCR5-1625	+	AGAAGACUAAGAGGUAGUUU	20	5434
  CCR5-2561	+	AAGAAGACUAAGAGGUAGUUU	21	5435
  CCR5-2562	+	GAAGAAGACUAAGAGGUAGUUU	22	5436
  CCR5-2563	+	GGAAGAAGACUAAGAGGUAGUUU	23	5437
  CCR5-2564	+	UGGAAGAAGACUAAGAGGUAGUUU	24	5438
  CCR5-2147	−	UCUUUACCAGAUCUCAAA	18	5439
  CCR5-2148	−	AUCUUUACCAGAUCUCAAA	19	5440
  CCR5-2149	−	CAUCUUUACCAGAUCUCAAA	20	5441
  CCR5-2150	−	UCAUCUUUACCAGAUCUCAAA	21	5442
  CCR5-2151	−	AUCAUCUUUACCAGAUCUCAAA	22	5443
  CCR5-2152	−	AAUCAUCUUUACCAGAUCUCAAA	23	5444
  CCR5-2153	−	GAAUCAUCUUUACCAGAUCUCAAA	24	5445
  CCR5-2565	−	CUUGUGACACGGACUCAA	18	5446
  CCR5-2566	−	GCUUGUGACACGGACUCAA	19	5447
  CCR5-963	−	GGCUUGUGACACGGACUCAA	20	5448
  CCR5-2567	−	GGGCUUGUGACACGGACUCAA	21	5449
  CCR5-2568	−	UGGGCUUGUGACACGGACUCAA	22	5450
  CCR5-2569	−	GUGGGCUUGUGACACGGACUCAA	23	5451
  CCR5-2570	−	UGUGGGCUUGUGACACGGACUCAA	24	5452
  CCR5-2571	−	CUCUGCUUCGGUGUCGAA	18	5453
  CCR5-2572	−	ACUCUGCUUCGGUGUCGAA	19	5454
  CCR5-931	−	AACUCUGCUUCGGUGUCGAA	20	5455
  CCR5-2573	−	AAACUCUGCUUCGGUGUCGAA	21	5456
  CCR5-2574	−	AAAACUCUGCUUCGGUGUCGAA	22	5457
  CCR5-2575	−	AAAAACUCUGCUUCGGUGUCGAA	23	5458
  CCR5-2576	−	UAAAAACUCUGCUUCGGUGUCGAA	24	5459
  CCR5-2577	−	CAGUUUACACCCGAUCCA	18	5460
  CCR5-2578	−	UCAGUUUACACCCGAUCCA	19	5461
  CCR5-955	−	CUCAGUUUACACCCGAUCCA	20	5462
  CCR5-2579	−	GCUCAGUUUACACCCGAUCCA	21	5463
  CCR5-2580	−	AGCUCAGUUUACACCCGAUCCA	22	5464
  CCR5-2581	−	AAGCUCAGUUUACACCCGAUCCA	23	5465
  CCR5-2582	−	CAAGCUCAGUUUACACCCGAUCCA	24	5466
  CCR5-2583	−	AAAUGAGAAGAAGAGGCA	18	5467
  CCR5-2584	−	GAAAUGAGAAGAAGAGGCA	19	5468
  CCR5-935	−	CGAAAUGAGAAGAAGAGGCA	20	5469
  CCR5-2585	−	UCGAAAUGAGAAGAAGAGGCA	21	5470
  CCR5-2586	−	GUCGAAAUGAGAAGAAGAGGCA	22	5471
  CCR5-2587	−	UGUCGAAAUGAGAAGAAGAGGCA	23	5472
  CCR5-2588	−	GUGUCGAAAUGAGAAGAAGAGGCA	24	5473
  CCR5-2589	−	CCAGCAAGAGGCUCCCGA	18	5474
  CCR5-2590	−	UCCAGCAAGAGGCUCCCGA	19	5475
  CCR5-954	−	UUCCAGCAAGAGGCUCCCGA	20	5476
  CCR5-2591	−	UUUCCAGCAAGAGGCUCCCGA	21	5477
  CCR5-2592	−	UUUUCCAGCAAGAGGCUCCCGA	22	5478
  CCR5-2593	−	AUUUUCCAGCAAGAGGCUCCCGA	23	5479
  CCR5-2594	−	UAUUUUCCAGCAAGAGGCUCCCGA	24	5480
  CCR5-2595	−	ACCAAGCUAUGCAGGUGA	18	5481
  CCR5-2596	−	GACCAAGCUAUGCAGGUGA	19	5482
  CCR5-943	−	GGACCAAGCUAUGCAGGUGA	20	5483
  CCR5-2597	−	UGGACCAAGCUAUGCAGGUGA	21	5484
  CCR5-2598	−	UUGGACCAAGCUAUGCAGGUGA	22	5485
  CCR5-2599	−	GUUGGACCAAGCUAUGCAGGUGA	23	5486
  CCR5-2600	−	GGUUGGACCAAGCUAUGCAGGUGA	24	5487
  CCR5-2601	−	AGUUUACACCCGAUCCAC	18	5488
  CCR5-2602	−	CAGUUUACACCCGAUCCAC	19	5489
  CCR5-178	−	UCAGUUUACACCCGAUCCAC	20	5490
  CCR5-2603	−	CUCAGUUUACACCCGAUCCAC	21	5491
  CCR5-2604	−	GCUCAGUUUACACCCGAUCCAC	22	5492
  CCR5-2605	−	AGCUCAGUUUACACCCGAUCCAC	23	5493
  CCR5-2606	−	AAGCUCAGUUUACACCCGAUCCAC	24	5494
  CCR5-2607	−	UAUCUGUGGGCUUGUGAC	18	5495
  CCR5-2608	−	AUAUCUGUGGGCUUGUGAC	19	5496
  CCR5-962	−	AAUAUCUGUGGGCUUGUGAC	20	5497
  CCR5-2609	−	AAAUAUCUGUGGGCUUGUGAC	21	5498
  CCR5-2610	−	GAAAUAUCUGUGGGCUUGUGAC	22	5499
  CCR5-2611	−	GGAAAUAUCUGUGGGCUUGUGAC	23	5500
  CCR5-2612	−	AGGAAAUAUCUGUGGGCUUGUGAC	24	5501
  CCR5-2613	−	GUCAUGGUCAUCUGCUAC	18	5502
  CCR5-2614	−	UGUCAUGGUCAUCUGCUAC	19	5503
  CCR5-927	−	UUGUCAUGGUCAUCUGCUAC	20	5504
  CCR5-2615	−	CUUGUCAUGGUCAUCUGCUAC	21	5505
  CCR5-2616	−	GCUUGUCAUGGUCAUCUGCUAC	22	5506
  CCR5-2617	−	UGCUUGUCAUGGUCAUCUGCUAC	23	5507
  CCR5-2618	−	CUGCUUGUCAUGGUCAUCUGCUAC	24	5508
  CCR5-2619	−	GCUGUUCUAUUUUCCAGC	18	5509
  CCR5-2620	−	UGCUGUUCUAUUUUCCAGC	19	5510
  CCR5-952	−	AUGCUGUUCUAUUUUCCAGC	20	5511
  CCR5-2621	−	AAUGCUGUUCUAUUUUCCAGC	21	5512
  CCR5-2622	−	AAAUGCUGUUCUAUUUUCCAGC	22	5513
  CCR5-2623	−	CAAAUGCUGUUCUAUUUUCCAGC	23	5514
  CCR5-2624	−	GCAAAUGCUGUUCUAUUUUCCAGC	24	5515
  CCR5-2625	−	CCCGAUCCACUGGGGAGC	18	5516
  CCR5-2626	−	ACCCGAUCCACUGGGGAGC	19	5517
  CCR5-181	−	CACCCGAUCCACUGGGGAGC	20	5518
  CCR5-2627	−	ACACCCGAUCCACUGGGGAGC	21	5519
  CCR5-2628	−	UACACCCGAUCCACUGGGGAGC	22	5520
  CCR5-2629	−	UUACACCCGAUCCACUGGGGAGC	23	5521
  CCR5-2630	−	UUUACACCCGAUCCACUGGGGAGC	24	5522
  CCR5-2631	−	ACAUUAAAGAUAGUCAUC	18	5523
  CCR5-2632	−	GACAUUAAAGAUAGUCAUC	19	5524
  CCR5-925	−	AGACAUUAAAGAUAGUCAUC	20	5525
  CCR5-2633	−	CAGACAUUAAAGAUAGUCAUC	21	5526
  CCR5-2634	−	CCAGACAUUAAAGAUAGUCAUC	22	5527
  CCR5-2635	−	UCCAGACAUUAAAGAUAGUCAUC	23	5528
  CCR5-2636	−	UUCCAGACAUUAAAGAUAGUCAUC	24	5529
  CCR5-2637	−	UGCAGGUGACAGAGACUC	18	5530
  CCR5-2638	−	AUGCAGGUGACAGAGACUC	19	5531
  CCR5-944	−	UAUGCAGGUGACAGAGACUC	20	5532
  CCR5-2639	−	CUAUGCAGGUGACAGAGACUC	21	5533
  CCR5-2640	−	GCUAUGCAGGUGACAGAGACUC	22	5534
  CCR5-2641	−	AGCUAUGCAGGUGACAGAGACUC	23	5535
  CCR5-2642	−	AAGCUAUGCAGGUGACAGAGACUC	24	5536
  CCR5-2643	−	UUUUCCAGCAAGAGGCUC	18	5537
  CCR5-2644	−	AUUUUCCAGCAAGAGGCUC	19	5538
  CCR5-953	−	UAUUUUCCAGCAAGAGGCUC	20	5539
  CCR5-2645	−	CUAUUUUCCAGCAAGAGGCUC	21	5540
  CCR5-2646	−	UCUAUUUUCCAGCAAGAGGCUC	22	5541
  CCR5-2647	−	UUCUAUUUUCCAGCAAGAGGCUC	23	5542
  CCR5-2648	−	GUUCUAUUUUCCAGCAAGAGGCUC	24	5543
  CCR5-2649	−	UACAACAUUGUCCUUCUC	18	5544
  CCR5-2650	−	CUACAACAUUGUCCUUCUC	19	5545
  CCR5-938	−	CCUACAACAUUGUCCUUCUC	20	5546
  CCR5-2651	−	CCCUACAACAUUGUCCUUCUC	21	5547
  CCR5-2652	−	UCCCUACAACAUUGUCCUUCUC	22	5548
  CCR5-2653	−	CUCCCUACAACAUUGUCCUUCUC	23	5549
  CCR5-2654	−	GCUCCCUACAACAUUGUCCUUCUC	24	5550
  CCR5-2655	−	AUCAUCUAUGCCUUUGUC	18	5551
  CCR5-2656	−	CAUCAUCUAUGCCUUUGUC	19	5552
  CCR5-175	−	CCAUCAUCUAUGCCUUUGUC	20	5553
  CCR5-2657	−	CCCAUCAUCUAUGCCUUUGUC	21	5554
  CCR5-2658	−	CCCCAUCAUCUAUGCCUUUGUC	22	5555
  CCR5-2659	−	ACCCCAUCAUCUAUGCCUUUGUC	23	5556
  CCR5-2660	−	AACCCCAUCAUCUAUGCCUUUGUC	24	5557
  CCR5-2661	−	UACAGUCAGUAUCAAUUC	18	5558
  CCR5-2662	−	AUACAGUCAGUAUCAAUUC	19	5559
  CCR5-152	−	CAUACAGUCAGUAUCAAUUC	20	5560
  CCR5-2663	−	CCAUACAGUCAGUAUCAAUUC	21	5561
  CCR5-2664	−	UCCAUACAGUCAGUAUCAAUUC	22	5562
  CCR5-2665	−	UUCCAUACAGUCAGUAUCAAUUC	23	5563
  CCR5-2666	−	UUUCCAUACAGUCAGUAUCAAUUC	24	5564
  CCR5-2667	−	CUUCUCCUGAACACCUUC	18	5565
  CCR5-2668	−	CCUUCUCCUGAACACCUUC	19	5566
  CCR5-939	−	UCCUUCUCCUGAACACCUUC	20	5567
  CCR5-2669	−	GUCCUUCUCCUGAACACCUUC	21	5568
  CCR5-2670	−	UGUCCUUCUCCUGAACACCUUC	22	5569
  CCR5-2671	−	UUGUCCUUCUCCUGAACACCUUC	23	5570
  CCR5-2672	−	AUUGUCCUUCUCCUGAACACCUUC	24	5571
  CCR5-2673	−	CGGUGUCGAAAUGAGAAG	18	5572
  CCR5-2674	−	UCGGUGUCGAAAUGAGAAG	19	5573
  CCR5-934	−	UUCGGUGUCGAAAUGAGAAG	20	5574
  CCR5-2675	−	CUUCGGUGUCGAAAUGAGAAG	21	5575
  CCR5-2676	−	GCUUCGGUGUCGAAAUGAGAAG	22	5576
  CCR5-2677	−	UGCUUCGGUGUCGAAAUGAGAAG	23	5577
  CCR5-2678	−	CUGCUUCGGUGUCGAAAUGAGAAG	24	5578
  CCR5-2679	−	ACCCGAUCCACUGGGGAG	18	5579
  CCR5-2680	−	CACCCGAUCCACUGGGGAG	19	5580
  CCR5-959	−	ACACCCGAUCCACUGGGGAG	20	5581
  CCR5-2681	−	UACACCCGAUCCACUGGGGAG	21	5582
  CCR5-2682	−	UUACACCCGAUCCACUGGGGAG	22	5583
  CCR5-2683	−	UUUACACCCGAUCCACUGGGGAG	23	5584
  CCR5-2684	−	GUUUACACCCGAUCCACUGGGGAG	24	5585
  CCR5-2685	−	CUUCGGUGUCGAAAUGAG	18	5586
  CCR5-2686	−	GCUUCGGUGUCGAAAUGAG	19	5587
  CCR5-933	−	UGCUUCGGUGUCGAAAUGAG	20	5588
  CCR5-2687	−	CUGCUUCGGUGUCGAAAUGAG	21	5589
  CCR5-2688	−	UCUGCUUCGGUGUCGAAAUGAG	22	5590
  CCR5-2689	−	CUCUGCUUCGGUGUCGAAAUGAG	23	5591
  CCR5-2690	−	ACUCUGCUUCGGUGUCGAAAUGAG	24	5592
  CCR5-2691	−	UCAUCUAUGCCUUUGUCG	18	5593
  CCR5-2692	−	AUCAUCUAUGCCUUUGUCG	19	5594
  CCR5-176	−	CAUCAUCUAUGCCUUUGUCG	20	5595
  CCR5-2693	−	CCAUCAUCUAUGCCUUUGUCG	21	5596
  CCR5-2694	−	CCCAUCAUCUAUGCCUUUGUCG	22	5597
  CCR5-2695	−	CCCCAUCAUCUAUGCCUUUGUCG	23	5598
  CCR5-2696	−	ACCCCAUCAUCUAUGCCUUUGUCG	24	5599
  CCR5-2697	−	UGCAGUAGCUCUAACAGG	18	5600
  CCR5-2698	−	UUGCAGUAGCUCUAACAGG	19	5601
  CCR5-942	−	AUUGCAGUAGCUCUAACAGG	20	5602
  CCR5-2699	−	AAUUGCAGUAGCUCUAACAGG	21	5603
  CCR5-2700	−	UAAUUGCAGUAGCUCUAACAGG	22	5604
  CCR5-2701	−	AUAAUUGCAGUAGCUCUAACAGG	23	5605
  CCR5-2702	−	AAUAAUUGCAGUAGCUCUAACAGG	24	5606
  CCR5-2703	−	AUCUAUGCCUUUGUCGGG	18	5607
  CCR5-2704	−	CAUCUAUGCCUUUGUCGGG	19	5608
  CCR5-950	−	UCAUCUAUGCCUUUGUCGGG	20	5609
  CCR5-2705	−	AUCAUCUAUGCCUUUGUCGGG	21	5610
  CCR5-2706	−	CAUCAUCUAUGCCUUUGUCGGG	22	5611
  CCR5-2707	−	CCAUCAUCUAUGCCUUUGUCGGG	23	5612
  CCR5-2708	−	CCCAUCAUCUAUGCCUUUGUCGGG	24	5613
  CCR5-2709	−	UUUACACCCGAUCCACUG	18	5614
  CCR5-2710	−	GUUUACACCCGAUCCACUG	19	5615
  CCR5-180	−	AGUUUACACCCGAUCCACUG	20	5616
  CCR5-2711	−	CAGUUUACACCCGAUCCACUG	21	5617
  CCR5-2712	−	UCAGUUUACACCCGAUCCACUG	22	5618
  CCR5-2713	−	CUCAGUUUACACCCGAUCCACUG	23	5619
  CCR5-2714	−	GCUCAGUUUACACCCGAUCCACUG	24	5620
  CCR5-2715	−	AAAAACUCUGCUUCGGUG	18	5621
  CCR5-2716	−	UAAAAACUCUGCUUCGGUG	19	5622
  CCR5-930	−	CUAAAAACUCUGCUUCGGUG	20	5623
  CCR5-2717	−	CCUAAAAACUCUGCUUCGGUG	21	5624
  CCR5-2718	−	UCCUAAAAACUCUGCUUCGGUG	22	5625
  CCR5-2719	−	AUCCUAAAAACUCUGCUUCGGUG	23	5626
  CCR5-2720	−	AAUCCUAAAAACUCUGCUUCGGUG	24	5627
  CCR5-2721	−	CCAUCAUCUAUGCCUUUG	18	5628
  CCR5-2722	−	CCCAUCAUCUAUGCCUUUG	19	5629
  CCR5-946	−	CCCCAUCAUCUAUGCCUUUG	20	5630
  CCR5-2723	−	ACCCCAUCAUCUAUGCCUUUG	21	5631
  CCR5-2724	−	AACCCCAUCAUCUAUGCCUUUG	22	5632
  CCR5-2725	−	CAACCCCAUCAUCUAUGCCUUUG	23	5633
  CCR5-2726	−	UCAACCCCAUCAUCUAUGCCUUUG	24	5634
  CCR5-2727	−	CUGCUUCGGUGUCGAAAU	18	5635
  CCR5-2728	−	UCUGCUUCGGUGUCGAAAU	19	5636
  CCR5-932	−	CUCUGCUUCGGUGUCGAAAU	20	5637
  CCR5-2729	−	ACUCUGCUUCGGUGUCGAAAU	21	5638
  CCR5-2730	−	AACUCUGCUUCGGUGUCGAAAU	22	5639
  CCR5-2731	−	AAACUCUGCUUCGGUGUCGAAAU	23	5640
  CCR5-2732	−	AAAACUCUGCUUCGGUGUCGAAAU	24	5641
  CCR5-2733	−	GUUUACACCCGAUCCACU	18	5642
  CCR5-2734	−	AGUUUACACCCGAUCCACU	19	5643
  CCR5-179	−	CAGUUUACACCCGAUCCACU	20	5644
  CCR5-2735	−	UCAGUUUACACCCGAUCCACU	21	5645
  CCR5-2736	−	CUCAGUUUACACCCGAUCCACU	22	5646
  CCR5-2737	−	GCUCAGUUUACACCCGAUCCACU	23	5647
  CCR5-2738	−	AGCUCAGUUUACACCCGAUCCACU	24	5648
  CCR5-2739	−	UCAUGGUCAUCUGCUACU	18	5649
  CCR5-2740	−	GUCAUGGUCAUCUGCUACU	19	5650
  CCR5-158	−	UGUCAUGGUCAUCUGCUACU	20	5651
  CCR5-2741	−	UUGUCAUGGUCAUCUGCUACU	21	5652
  CCR5-2742	−	CUUGUCAUGGUCAUCUGCUACU	22	5653
  CCR5-2743	−	GCUUGUCAUGGUCAUCUGCUACU	23	5654
  CCR5-2744	−	UGCUUGUCAUGGUCAUCUGCUACU	24	5655
  CCR5-2745	−	AAGAAGAGGCACAGGGCU	18	5656
  CCR5-2746	−	GAAGAAGAGGCACAGGGCU	19	5657
  CCR5-936	−	AGAAGAAGAGGCACAGGGCU	20	5658
  CCR5-2747	−	GAGAAGAAGAGGCACAGGGCU	21	5659
  CCR5-2748	−	UGAGAAGAAGAGGCACAGGGCU	22	5660
  CCR5-2749	−	AUGAGAAGAAGAGGCACAGGGCU	23	5661
  CCR5-2750	−	AAUGAGAAGAAGAGGCACAGGGCU	24	5662
  CCR5-2751	−	CAUUAAAGAUAGUCAUCU	18	5663
  CCR5-2752	−	ACAUUAAAGAUAGUCAUCU	19	5664
  CCR5-153	−	GACAUUAAAGAUAGUCAUCU	20	5665
  CCR5-2753	−	AGACAUUAAAGAUAGUCAUCU	21	5666
  CCR5-2754	−	CAGACAUUAAAGAUAGUCAUCU	22	5667
  CCR5-2755	−	CCAGACAUUAAAGAUAGUCAUCU	23	5668
  CCR5-2756	−	UCCAGACAUUAAAGAUAGUCAUCU	24	5669
  CCR5-2757	−	GGGGAGCAGGAAAUAUCU	18	5670
  CCR5-2758	−	UGGGGAGCAGGAAAUAUCU	19	5671
  CCR5-961	−	CUGGGGAGCAGGAAAUAUCU	20	5672
  CCR5-2759	−	ACUGGGGAGCAGGAAAUAUCU	21	5673
  CCR5-2760	−	CACUGGGGAGCAGGAAAUAUCU	22	5674
  CCR5-2761	−	CCACUGGGGAGCAGGAAAUAUCU	23	5675
  CCR5-2762	−	UCCACUGGGGAGCAGGAAAUAUCU	24	5676
  CCR5-2763	−	CAUCAUCUAUGCCUUUGU	18	5677
  CCR5-2764	−	CCAUCAUCUAUGCCUUUGU	19	5678
  CCR5-174	−	CCCAUCAUCUAUGCCUUUGU	20	5679
  CCR5-2765	−	CCCCAUCAUCUAUGCCUUUGU	21	5680
  CCR5-2766	−	ACCCCAUCAUCUAUGCCUUUGU	22	5681
  CCR5-2767	−	AACCCCAUCAUCUAUGCCUUUGU	23	5682
  CCR5-2768	−	CAACCCCAUCAUCUAUGCCUUUGU	24	5683
  CCR5-2769	−	AUACAGUCAGUAUCAAUU	18	5684
  CCR5-2770	−	CAUACAGUCAGUAUCAAUU	19	5685
  CCR5-922	−	CCAUACAGUCAGUAUCAAUU	20	5686
  CCR5-2771	−	UCCAUACAGUCAGUAUCAAUU	21	5687
  CCR5-2772	−	UUCCAUACAGUCAGUAUCAAUU	22	5688
  CCR5-2773	−	UUUCCAUACAGUCAGUAUCAAUU	23	5689
  CCR5-2774	−	UUUUCCAUACAGUCAGUAUCAAUU	24	5690
  CCR5-2775	−	GAUUGUUUAUUUUCUCUU	18	5691
  CCR5-2776	−	UGAUUGUUUAUUUUCUCUU	19	5692
  CCR5-937	−	AUGAUUGUUUAUUUUCUCUU	20	5693
  CCR5-2777	−	CAUGAUUGUUUAUUUUCUCUU	21	5694
  CCR5-2778	−	UCAUGAUUGUUUAUUUUCUCUU	22	5695
  CCR5-2779	−	AUCAUGAUUGUUUAUUUUCUCUU	23	5696
  CCR5-2780	−	CAUCAUGAUUGUUUAUUUUCUCUU	24	5697
  CCR5-2781	−	CUUUGUCGGGGAGAAGUU	18	5698
  CCR5-2782	−	CCUUUGUCGGGGAGAAGUU	19	5699
  CCR5-951	−	GCCUUUGUCGGGGAGAAGUU	20	5700
  CCR5-2783	−	UGCCUUUGUCGGGGAGAAGUU	21	5701
  CCR5-2784	−	AUGCCUUUGUCGGGGAGAAGUU	22	5702
  CCR5-2785	−	UAUGCCUUUGUCGGGGAGAAGUU	23	5703
  CCR5-2786	−	CUAUGCCUUUGUCGGGGAGAAGUU	24	5704

• Table 4A provides exemplary targeting domains for knocking out the CCR5 gene selected according to the first tier parameters. The targeting domains bind within the first 500 bp of the coding sequence (e.g., with 500 bp downstream from the start codon) and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 4A
  1st Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-2787	−	UGCACAGGGUGGAACAA	17	5705
  CCR5-1824	+	GGCUGCGAUUUGCUUCA	17	5706
  CCR5-1821	+	GACGACAGCCAGGUACC	17	5707
  CCR5-1823	+	CGGAGGCAGGAGGCGGG	17	5708
  CCR5-1825	+	UGUAUAAUAAUUGAUGU	17	5709
  CCR5-2788	−	GCUGUCGUCCAUGCUGU	17	5710
  CCR5-2789	−	UGACAGGGCUCUAUUUU	17	5711
  CCR5-2790	−	UUAUGCACAGGGUGGAACAA	20	5712
  CCR5-1819	+	GCGGGCUGCGAUUUGCUUCA	20	5713
  CCR5-1816	+	AUGGACGACAGCCAGGUACC	20	5714
  CCR5-1818	+	GAGCGGAGGCAGGAGGCGGG	20	5715
  CCR5-1820	+	CGAUGUAUAAUAAUUGAUGU	20	5716
  CCR5-2791	−	UCUUGACAGGGCUCUAUUUU	20	5717

• Table 4B provides exemplary targeting domains for knocking out the CCR5 gene selected according to the second tier parameters. The targeting domains bind within the first 500 bp of the coding sequence (e.g., with 500 bp downstream from the start codon). It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 4B
  2nd Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-2792	+	UUUUUGAGAUCUGGUAA	17	5718
  CCR5-1822	+	UGUCAGGAGGAUGAUGA	17	5719
  CCR5-2793	+	GCAGGAGGCGGGCUGCG	17	5720
  CCR5-2794	+	ACCCCAAAGGUGACCGU	17	5721
  CCR5-2795	+	UUCUUUUUGAGAUCUGGUAA	20	5722
  CCR5-1817	+	GAUUGUCAGGAGGAUGAUGA	20	5723
  CCR5-2796	+	GAGGCAGGAGGCGGGCUGCG	20	5724
  CCR5-2797	+	ACCACCCCAAAGGUGACCGU	20	5725
  CCR5-2798	−	CUGGCUGUCGUCCAUGCUGU	20	5726

• Table 4C provides exemplary targeting domains for knocking out the CCR5 gene selected according to the third tier parameters. The targeting domains fall in the coding sequence of the gene, downstream of the first 500 bp of coding sequence (e.g., anywhere from +500 (relative to the start codon) to the stop codon of the gene. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis Cas9 molecule that generates a double stranded break (Cas9 nuclease) or a single-stranded break (Cas9 nickase).

• TABLE 4C
  3rd Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-2799	−	CUCGGGAAUCCUAAAAA	17	5727
  CCR5-1771	+	AGUGGAUCGGGUGUAAA	17	5728
  CCR5-2792	+	UUUUUGAGAUCUGGUAA	17	5729
  CCR5-1841	−	GAGGCUUAUCUUCACCA	17	5730
  CCR5-2800	+	UGCAGAAGCGUUUGGCA	17	5731
  CCR5-2801	−	UCCAAAAGCACAUUGCC	17	5732
  CCR5-2802	−	CUUGGGGCUGGUCCUGC	17	5733
  CCR5-2803	+	AGAGUCUCUGUCACCUG	17	5734
  CCR5-2804	−	GAAGAGGCACAGGGCUG	17	5735
  CCR5-1250	−	GGGAGCAGGAAAUAUCU	17	5736
  CCR5-1863	+	ACACCGAAGCAGAGUUU	17	5737
  CCR5-2805	−	CUACUCGGGAAUCCUAAAAA	20	5738
  CCR5-1613	+	CCCAGUGGAUCGGGUGUAAA	20	5739
  CCR5-2795	+	UUCUUUUUGAGAUCUGGUAA	20	5740
  CCR5-1826	−	UGUGAGGCUUAUCUUCACCA	20	5741
  CCR5-2806	+	AUUUGCAGAAGCGUUUGGCA	20	5742
  CCR5-2807	−	UCUUCCAAAAGCACAUUGCC	20	5743
  CCR5-2808	−	CAUCUUGGGGCUGGUCCUGC	20	5744
  CCR5-2809	+	CCAAGAGUCUCUGUCACCUG	20	5745
  CCR5-2810	−	GAAGAAGAGGCACAGGGCUG	20	5746
  CCR5-961	−	CUGGGGAGCAGGAAAUAUCU	20	5747
  CCR5-1859	+	UCGACACCGAAGCAGAGUUU	20	5748

• Table 5A provides exemplary targeting domains for knocking down the CCR5 gene selected according to the first tier parameters. The targeting domains bind within 500 bp (e.g., upstream or downstream) of a transcription start site (TSS) and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 5A
  1st Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-2811	+	CUCAGAAGCUAACUAAC	17	2217
  CCR5-2812	+	UUACGGGCUUUUCUCAC	17	2218
  CCR5-2813	+	UGAGAGGUUACUUACCG	17	2219
  CCR5-2814	+	AGAAUAGAUCUCUGGUCUGA	20	2220
  CCR5-2815	+	CUGGUCUGAAGGUUUAUUUA	20	2221
  CCR5-2816	+	CAUCUCAGAAGCUAACUAAC	20	2222
  CCR5-2817	+	UGGUCUGAAGGUUUAUUUAC	20	2223
  CCR5-2818	−	CCCCUACAAGAAACUCUCCC	20	2224
  CCR5-2819	−	GAUAGGGGAUACGGGGAGAG	20	2225
  CCR5-2820	+	CCGGGGAGAGUUUCUUGUAG	20	2226
  CCR5-2821	+	AGCUGAGAGGUUACUUACCG	20	2227
  CCR5-2822	+	AAGAUAAUUGUAUGAGCACU	20	2228
  CCR5-2823	−	UCCCCCUCUACAUUUAAAGU	20	2229

• Table 5B provides exemplary targeting domains for knocking down the CCR5 gene selected according to the second tier parameters. The targeting domains bind within 500 bp (e.g., upstream or downstream) of a transcription start site (TSS). It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNA may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 5B
  2nd Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-2824	−	GGGAGAGUGGAGAAAAA	17	2230
  CCR5-2825	−	GGGGAGAGUGGAGAAAA	17	2231
  CCR5-2826	−	UCUUUAAGAUAAGGAAA	17	2232
  CCR5-2827	+	UCAACAGUAAGGCUAAA	17	2233
  CCR5-2828	−	GAGUGAAAGACUUUAAA	17	2234
  CCR5-2829	−	AUCUUUAAGAUAAGGAA	17	2235
  CCR5-2830	+	AGUUUCUUGUAGGGGAA	17	2236
  CCR5-2831	+	GAAAAUAUAAAGAAUAA	17	2237
  CCR5-2832	−	UGAGUGAAAGACUUUAA	17	2238
  CCR5-2833	−	GAGAAAAAGGGGACACA	17	2239
  CCR5-2834	+	AUUUGUACAAGAUCACA	17	2240
  CCR5-2835	−	UUGGAAUGAGUUUCAGA	17	2241
  CCR5-2836	+	AGGCAUCUCACUGGAGA	17	2242
  CCR5-2837	+	CCAACUUUAAAUGUAGA	17	2243
  CCR5-2838	+	CUGUUUCUUUUGAAGGA	17	2244
  CCR5-2839	+	AUAGAUCUCUGGUCUGA	17	2245
  CCR5-2840	+	AUCAUUAAGUGUAUUGA	17	2246
  CCR5-2841	+	AAUGCUGUUUCUUUUGA	17	2247
  CCR5-2842	−	AUAUAAUCUUUAAGAUA	17	2248
  CCR5-2843	−	GGGUGGGAUAGGGGAUA	17	2249
  CCR5-2844	−	GGGGUUGGGGUGGGAUA	17	2250
  CCR5-2845	−	AAUCUUAUCUUCUGCUA	17	2251
  CCR5-2846	+	UUGCCAAAUGUCUUCUA	17	2252
  CCR5-2847	+	AGGGCUUUUCAACAGUA	17	2253
  CCR5-2848	+	CUUUCUUUUGAGAGGUA	17	2254
  CCR5-2849	+	GGGGAGAGUUUCUUGUA	17	2255
  CCR5-2850	+	GUCUGAAGGUUUAUUUA	17	2256
  CCR5-2851	−	GGAGAAAAAGGGGACAC	17	2257
  CCR5-2852	+	GAUUUGUACAAGAUCAC	17	2258
  CCR5-2853	+	UUCAGAAGGCAUCUCAC	17	2259
  CCR5-2854	−	GGUGGGAUAGGGGAUAC	17	2260
  CCR5-2855	+	GCUGAGAGGUUACUUAC	17	2261
  CCR5-2856	+	UCUGAAGGUUUAUUUAC	17	2262
  CCR5-2857	−	UGAGUAAAAGACUUUAC	17	2263
  CCR5-2858	+	CUGAGAGGUUACUUACC	17	2264
  CCR5-2859	−	CUACAAGAAACUCUCCC	17	2265
  CCR5-2860	+	AAUGUAGAGGGGGAUCC	17	2266
  CCR5-2861	−	GGGUUAAUGUGAAGUCC	17	2267
  CCR5-2862	−	GAUUUGCACAGCUCAUC	17	2268
  CCR5-2863	+	GCUAGAGAAUAGAUCUC	17	2269
  CCR5-2864	+	GGAUGUCUCAGCUCUUC	17	2270
  CCR5-2865	−	GGAGAGUGGAGAAAAAG	17	2271
  CCR5-2866	−	AGGGGAUACGGGGAGAG	17	2272
  CCR5-2867	+	CAACUUUAAAUGUAGAG	17	2273
  CCR5-2868	+	AAGGCAUCUCACUGGAG	17	2274
  CCR5-2869	+	CAGGCCAAGCAGCUGAG	17	2275
  CCR5-2870	+	CAAAUCUUUCUUUUGAG	17	2276
  CCR5-2871	−	GGGUUGGGGUGGGAUAG	17	2277
  CCR5-2872	+	ACCAACUUUAAAUGUAG	17	2278
  CCR5-2873	−	UAACAGAUUCUGUGUAG	17	2279
  CCR5-2874	+	GGGAGAGUUUCUUGUAG	17	2280
  CCR5-2875	−	GUGGGAUAGGGGAUACG	17	2281
  CCR5-2876	+	GCUGUUUCUUUUGAAGG	17	2282
  CCR5-2877	+	AACUUUAAAUGUAGAGG	17	2283
  CCR5-2878	+	UUUCUUUUGAAGGAGGG	17	2284
  CCR5-2879	−	CUGUGUGGGGGUUGGGG	17	2285
  CCR5-2880	−	AGAACAAUAAUAUUGGG	17	2286
  CCR5-2881	−	GGUGAGCAUCUGUGUGG	17	2287
  CCR5-2882	−	UUUCUUUUACUAAAAUG	17	2288
  CCR5-2883	−	GGUGGUGAGCAUCUGUG	17	2289
  CCR5-2884	−	UGGUGAGCAUCUGUGUG	17	2290
  CCR5-2885	−	CAUCUGUGUGGGGGUUG	17	2291
  CCR5-2886	−	GGGGGUUGGGGUGGGAU	17	2292
  CCR5-2887	−	ACAGAGAACAAUAAUAU	17	2293
  CCR5-2888	+	UGCCAAAUGUCUUCUAU	17	2294
  CCR5-2889	+	AUAAUUGUAUGAGCACU	17	2295
  CCR5-2890	−	GUAACCUCUCAGCUGCU	17	2296
  CCR5-2891	−	ACAAAUCAUUUGCUUCU	17	2297
  CCR5-2892	+	AUAGACAGUAUAAAAGU	17	2298
  CCR5-2893	−	CCCUCUACAUUUAAAGU	17	2299
  CCR5-2894	−	UUAAAGUUGGUUUAAGU	17	2300
  CCR5-2895	−	AACAGAUUCUGUGUAGU	17	2301
  CCR5-2896	−	AGCAUCUGUGUGGGGGU	17	2302
  CCR5-2897	−	UGUGUGGGGGUUGGGGU	17	2303
  CCR5-2898	−	UUCUUUUACUAAAAUGU	17	2304
  CCR5-2899	−	GUGGUGAGCAUCUGUGU	17	2305
  CCR5-2900	+	CGGGGAGAGUUUCUUGU	17	2306
  CCR5-2901	−	AACCCAUAGAAGACAUU	17	2307
  CCR5-2902	−	CAGAGAACAAUAAUAUU	17	2308
  CCR5-2903	−	AGGAAAGGGUCACAGUU	17	2309
  CCR5-2904	−	GCAUCUGUGUGGGGGUU	17	2310
  CCR5-2905	−	ACGGGGAGAGUGGAGAAAAA	20	2311
  CCR5-2906	−	UACGGGGAGAGUGGAGAAAA	20	2312
  CCR5-2907	−	UAAUCUUUAAGAUAAGGAAA	20	2313
  CCR5-2908	+	UUUUCAACAGUAAGGCUAAA	20	2314
  CCR5-2909	−	UGUGAGUGAAAGACUUUAAA	20	2315
  CCR5-2910	−	AUAAUCUUUAAGAUAAGGAA	20	2316
  CCR5-2911	+	GAGAGUUUCUUGUAGGGGAA	20	2317
  CCR5-2912	+	UUAGAAAAUAUAAAGAAUAA	20	2318
  CCR5-2913	−	UUGUGAGUGAAAGACUUUAA	20	2319
  CCR5-2914	−	GUGGAGAAAAAGGGGACACA	20	2320
  CCR5-2915	+	AUGAUUUGUACAAGAUCACA	20	2321
  CCR5-2916	−	AGUUUGGAAUGAGUUUCAGA	20	2322
  CCR5-2917	+	AGAAGGCAUCUCACUGGAGA	20	2323
  CCR5-2918	+	AAACCAACUUUAAAUGUAGA	20	2324
  CCR5-2919	+	AUGCUGUUUCUUUUGAAGGA	20	2325
  CCR5-2920	+	UAAAUCAUUAAGUGUAUUGA	20	2326
  CCR5-2921	+	GGAAAUGCUGUUUCUUUUGA	20	2327
  CCR5-2922	−	AAAAUAUAAUCUUUAAGAUA	20	2328
  CCR5-2923	−	UUGGGGUGGGAUAGGGGAUA	20	2329
  CCR5-2924	−	GUGGGGGUUGGGGUGGGAUA	20	2330
  CCR5-2925	−	UGAAAUCUUAUCUUCUGCUA	20	2331
  CCR5-2926	+	UGUUUGCCAAAUGUCUUCUA	20	2332
  CCR5-2927	+	CACAGGGCUUUUCAACAGUA	20	2333
  CCR5-2928	+	AAUCUUUCUUUUGAGAGGUA	20	2334
  CCR5-2929	+	ACCGGGGAGAGUUUCUUGUA	20	2335
  CCR5-2930	−	AGUGGAGAAAAAGGGGACAC	20	2336
  CCR5-2931	+	AAUGAUUUGUACAAGAUCAC	20	2337
  CCR5-2932	+	AUAUUCAGAAGGCAUCUCAC	20	2338
  CCR5-2933	+	UAUUUACGGGCUUUUCUCAC	20	2339
  CCR5-2934	−	UGGGGUGGGAUAGGGGAUAC	20	2340
  CCR5-2935	+	GCAGCUGAGAGGUUACUUAC	20	2341
  CCR5-2936	−	AGAUGAGUAAAAGACUUUAC	20	2342
  CCR5-2937	+	CAGCUGAGAGGUUACUUACC	20	2343
  CCR5-2938	+	UUAAAUGUAGAGGGGGAUCC	20	2344
  CCR5-2939	−	ACAGGGUUAAUGUGAAGUCC	20	2345
  CCR5-2940	−	AUUGAUUUGCACAGCUCAUC	20	2346
  CCR5-2941	+	UAAGCUAGAGAAUAGAUCUC	20	2347
  CCR5-2942	+	AACGGAUGUCUCAGCUCUUC	20	2348
  CCR5-2943	−	CGGGGAGAGUGGAGAAAAAG	20	2349
  CCR5-2944	+	AACCAACUUUAAAUGUAGAG	20	2350
  CCR5-2945	+	CAGAAGGCAUCUCACUGGAG	20	2351
  CCR5-2946	+	UAACAGGCCAAGCAGCUGAG	20	2352
  CCR5-2947	+	CUGCAAAUCUUUCUUUUGAG	20	2353
  CCR5-2948	−	UGGGGGUUGGGGUGGGAUAG	20	2354
  CCR5-2949	+	UAAACCAACUUUAAAUGUAG	20	2355
  CCR5-2950	−	UUCUAACAGAUUCUGUGUAG	20	2356
  CCR5-2951	−	GGGGUGGGAUAGGGGAUACG	20	2357
  CCR5-2952	+	AAUGCUGUUUCUUUUGAAGG	20	2358
  CCR5-2953	+	ACCAACUUUAAAUGUAGAGG	20	2359
  CCR5-2954	+	CUGUUUCUUUUGAAGGAGGG	20	2360
  CCR5-2955	−	CAUCUGUGUGGGGGUUGGGG	20	2361
  CCR5-2956	−	CAGAGAACAAUAAUAUUGGG	20	2362
  CCR5-2957	−	GGUGGUGAGCAUCUGUGUGG	20	2363
  CCR5-2958	−	UAAUUUCUUUUACUAAAAUG	20	2364
  CCR5-2959	−	UUGGGUGGUGAGCAUCUGUG	20	2365
  CCR5-2960	−	GGGUGGUGAGCAUCUGUGUG	20	2366
  CCR5-2961	−	GAGCAUCUGUGUGGGGGUUG	20	2367
  CCR5-2962	−	UGUGGGGGUUGGGGUGGGAU	20	2368
  CCR5-2963	−	UUUACAGAGAACAAUAAUAU	20	2369
  CCR5-2964	+	GUUUGCCAAAUGUCUUCUAU	20	2370
  CCR5-2965	−	UAAGUAACCUCUCAGCUGCU	20	2371
  CCR5-2966	−	UGUACAAAUCAUUUGCUUCU	20	2372
  CCR5-2967	+	CAUAUAGACAGUAUAAAAGU	20	2373
  CCR5-2968	−	CAUUUAAAGUUGGUUUAAGU	20	2374
  CCR5-2969	−	UCUAACAGAUUCUGUGUAGU	20	2375
  CCR5-2970	−	GUGAGCAUCUGUGUGGGGGU	20	2376
  CCR5-2971	−	AUCUGUGUGGGGGUUGGGGU	20	2377
  CCR5-2972	−	AAUUUCUUUUACUAAAAUGU	20	2378
  CCR5-2973	−	UGGGUGGUGAGCAUCUGUGU	20	2379
  CCR5-2974	+	UACCGGGGAGAGUUUCUUGU	20	2380
  CCR5-2975	−	GGAAACCCAUAGAAGACAUU	20	2381
  CCR5-2976	−	UUACAGAGAACAAUAAUAUU	20	2382
  CCR5-2977	−	AUAAGGAAAGGGUCACAGUU	20	2383
  CCR5-2978	−	UGAGCAUCUGUGUGGGGGUU	20	2384

• Table 5C provides exemplary targeting domains for knocking down the CCR5 gene selected according to the third tier parameters. Within the additional 500 bp (e.g., upstream or downstream) of a transcription start site (TSS), e.g., extending to 1kb upstream and downstream of a TSS. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. pyogenes eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 5C
  3rd Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-2979	−	AGAGGGAAGCCUAAAAA	17	2385
  CCR5-2980	+	AUGCUUACUGGUUUGAA	17	2386
  CCR5-2981	−	GGAGUUUGAGACUCACA	17	2387
  CCR5-2982	+	UUUUUAUUCUAGAGCCA	17	2388
  CCR5-2983	−	GCCUAGUCUAAGGUGCA	17	2389
  CCR5-2984	−	UUUUAACUAUGGGCUCA	17	2390
  CCR5-2985	+	UUCUAGAGCCAAGGUCA	17	2391
  CCR5-2986	−	CUAAUAUAUCAGUUUCA	17	2392
  CCR5-2987	+	CUGGGUCCAGAAAAAGA	17	2393
  CCR5-2988	−	UUUUCCUCCAGACAAGA	17	2394
  CCR5-2989	−	GCUUGUGAUCUCUAAGA	17	2395
  CCR5-2990	+	GGUCACGGAAGCCCAGA	17	2396
  CCR5-2991	+	AAUGCUUACUGGUUUGA	17	2397
  CCR5-2992	−	CACAUGACAUAAGUAUA	17	2398
  CCR5-2993	−	CUAAAGAGUUUUAACUA	17	2399
  CCR5-2994	−	CUCAGCUGCCUAGUCUA	17	2400
  CCR5-2995	−	AAAAAUGAGCUUUUCUA	17	2401
  CCR5-2996	−	UAGUAUAUAAUUCUUUA	17	2402
  CCR5-2997	−	UCACGGGUGAGCUAAAC	17	2403
  CCR5-2998	+	AAAACUCUUUAGACAAC	17	2404
  CCR5-2999	−	GGGAGUUUGAGACUCAC	17	2405
  CCR5-3000	−	UUUAACUAUGGGCUCAC	17	2406
  CCR5-3001	+	UCCUCAUAAAUGCUUAC	17	2407
  CCR5-3002	−	CAUCUUUUUCUGGACCC	17	2408
  CCR5-3003	−	UCAUCUAUGACCUUCCC	17	2409
  CCR5-3004	+	AAUCCCCACUAAGAUCC	17	2410
  CCR5-3005	−	AGACUAGGCAAGACAGC	17	2411
  CCR5-3006	−	CCAGAUACAUAGGUGGC	17	2412
  CCR5-3007	−	UGCCUAGUCUAAGGUGC	17	2413
  CCR5-3008	+	UUCAGAUAGAUUAUAUC	17	2414
  CCR5-3009	+	CCUGCCACCUAUGUAUC	17	2415
  CCR5-3010	−	AGCCACAAGAUGCCCUC	17	2416
  CCR5-3011	+	AGGGCAUCUUGUGGCUC	17	2417
  CCR5-3012	−	GAAGUUGUGUCUAAGUC	17	2418
  CCR5-3013	+	UAGGCUUCCCUCUUGUC	17	2419
  CCR5-3014	+	AUGAAUGUCAUGCAUUC	17	2420
  CCR5-3015	−	AGUAUAUGGUCAAGUUC	17	2421
  CCR5-3016	−	GGUUUCCCAUCUUUUUC	17	2422
  CCR5-3017	−	UUUUUCCUCCAGACAAG	17	2423
  CCR5-3018	−	UGCCCCCAAUCCUACAG	17	2424
  CCR5-3019	+	AGGUCACGGAAGCCCAG	17	2425
  CCR5-3020	−	AAAAUGAGCUUUUCUAG	17	2426
  CCR5-3021	+	UGAAACUGAUAUAUUAG	17	2427
  CCR5-3022	−	UGGACCCAGGAUCUUAG	17	2428
  CCR5-3023	−	UAUGCCAGAUACAUAGG	17	2429
  CCR5-3024	+	GCUUCCCUCUUGUCUGG	17	2430
  CCR5-3025	−	AUGACAUUCAUCUGUGG	17	2431
  CCR5-3026	+	UGCCUCUGUAGGAUUGG	17	2432
  CCR5-3027	−	AUAUCAAGCUCUCUUGG	17	2433
  CCR5-3028	+	CAUAUACUUAUGUCAUG	17	2434
  CCR5-3029	−	ACCAGUAAGCAUUUAUG	17	2435
  CCR5-3030	−	UGCAUGACAUUCAUCUG	17	2436
  CCR5-3031	−	GACCCAGGAUCUUAGUG	17	2437
  CCR5-3032	−	ACUUCACAGAAAAUGUG	17	2438
  CCR5-3033	−	AUGACAACUCUUAAUUG	17	2439
  CCR5-3034	+	CUGCCUCUGUAGGAUUG	17	2440
  CCR5-3035	+	GCCCAGAGGGCAUCUUG	17	2441
  CCR5-3036	+	UUAGACACAACUUCUUG	17	2442
  CCR5-3037	+	CGUAAUUUUGCUGUUUG	17	2443
  CCR5-3038	−	UGUGAGGAUUUUACAAU	17	2444
  CCR5-3039	−	CACUAUGCCAGAUACAU	17	2445
  CCR5-3040	+	UGGGUCCAGAAAAAGAU	17	2446
  CCR5-3041	−	UAAAGAGUUUUAACUAU	17	2447
  CCR5-3042	−	CUGAACUUAAAUAGACU	17	2448
  CCR5-3043	+	UCCCUGCACCUUAGACU	17	2449
  CCR5-3044	−	CUGGGCUUCCGUGACCU	17	2450
  CCR5-3045	−	CAUCUAUGACCUUCCCU	17	2451
  CCR5-3046	+	AUCCCCACUAAGAUCCU	17	2452
  CCR5-3047	+	GAGGGCAUCUUGUGGCU	17	2453
  CCR5-3048	−	GCCACAAGAUGCCCUCU	17	2454
  CCR5-3049	−	GUCAUAUCAAGCUCUCU	17	2455
  CCR5-3050	+	UGAAUGUCAUGCAUUCU	17	2456
  CCR5-3051	−	UUUAUUAUAUUAUUUCU	17	2457
  CCR5-3052	−	UAAAAAUGAGCUUUUCU	17	2458
  CCR5-3053	−	GGACCCAGGAUCUUAGU	17	2459
  CCR5-3054	−	CAAGCUCUCUUGGCGGU	17	2460
  CCR5-3055	+	UAGACACAACUUCUUGU	17	2461
  CCR5-3056	+	UCUGCCUCUGUAGGAUU	17	2462
  CCR5-3057	+	UAGAGGAAAAUUUUAUU	17	2463
  CCR5-3058	−	UCUAGAAUAAAAAGCUU	17	2464
  CCR5-3059	−	UUAUUAUAUUAUUUCUU	17	2465
  CCR5-3060	+	CACGUAAUUUUGCUGUU	17	2466
  CCR5-3061	+	ACGUAAUUUUGCUGUUU	17	2467
  CCR5-3062	+	UAAUUUUGACCAUUUUU	17	2468
  CCR5-3063	−	ACAAGAGGGAAGCCUAAAAA	20	2469
  CCR5-3064	+	UAAAUGCUUACUGGUUUGAA	20	2470
  CCR5-3065	−	CAGGGAGUUUGAGACUCACA	20	2471
  CCR5-3066	+	AGCUUUUUAUUCUAGAGCCA	20	2472
  CCR5-3067	−	GCUGCCUAGUCUAAGGUGCA	20	2473
  CCR5-3068	−	GAGUUUUAACUAUGGGCUCA	20	2474
  CCR5-3069	+	UUAUUCUAGAGCCAAGGUCA	20	2475
  CCR5-3070	−	CCUCUAAUAUAUCAGUUUCA	20	2476
  CCR5-3071	+	AUCCUGGGUCCAGAAAAAGA	20	2477
  CCR5-3072	−	UCUUUUUCCUCCAGACAAGA	20	2478
  CCR5-3073	−	UUGGCUUGUGAUCUCUAAGA	20	2479
  CCR5-3074	+	CAAGGUCACGGAAGCCCAGA	20	2480
  CCR5-3075	+	AUAAAUGCUUACUGGUUUGA	20	2481
  CCR5-3076	−	UUCCACAUGACAUAAGUAUA	20	2482
  CCR5-3077	−	UGUCUAAAGAGUUUUAACUA	20	2483
  CCR5-3078	−	UCUCUCAGCUGCCUAGUCUA	20	2484
  CCR5-3079	−	AUUAAAAAUGAGCUUUUCUA	20	2485
  CCR5-3080	−	AGUUAGUAUAUAAUUCUUUA	20	2486
  CCR5-3081	−	GGCUCACGGGUGAGCUAAAC	20	2487
  CCR5-3082	+	GUUAAAACUCUUUAGACAAC	20	2488
  CCR5-3083	−	GCAGGGAGUUUGAGACUCAC	20	2489
  CCR5-3084	−	AGUUUUAACUAUGGGCUCAC	20	2490
  CCR5-3085	+	GAGUCCUCAUAAAUGCUUAC	20	2491
  CCR5-3086	−	UCCCAUCUUUUUCUGGACCC	20	2492
  CCR5-3087	−	UUGUCAUCUAUGACCUUCCC	20	2493
  CCR5-3088	+	GAAAAUCCCCACUAAGAUCC	20	2494
  CCR5-3089	−	AAUAGACUAGGCAAGACAGC	20	2495
  CCR5-3090	−	AUGCCAGAUACAUAGGUGGC	20	2496
  CCR5-3091	−	AGCUGCCUAGUCUAAGGUGC	20	2497
  CCR5-3092	+	AGCUUCAGAUAGAUUAUAUC	20	2498
  CCR5-3093	+	AAUCCUGCCACCUAUGUAUC	20	2499
  CCR5-3094	−	CCGAGCCACAAGAUGCCCUC	20	2500
  CCR5-3095	+	CAGAGGGCAUCUUGUGGCUC	20	2501
  CCR5-3096	−	CAAGAAGUUGUGUCUAAGUC	20	2502
  CCR5-3097	+	UUUUAGGCUUCCCUCUUGUC	20	2503
  CCR5-3098	+	CAGAUGAAUGUCAUGCAUUC	20	2504
  CCR5-3099	−	AUAAGUAUAUGGUCAAGUUC	20	2505
  CCR5-3100	−	ACAGGUUUCCCAUCUUUUUC	20	2506
  CCR5-3101	−	UUCUUUUUCCUCCAGACAAG	20	2507
  CCR5-3102	−	ACGUGCCCCCAAUCCUACAG	20	2508
  CCR5-3103	+	CCAAGGUCACGGAAGCCCAG	20	2509
  CCR5-3104	−	UUAAAAAUGAGCUUUUCUAG	20	2510
  CCR5-3105	+	CCAUGAAACUGAUAUAUUAG	20	2511
  CCR5-3106	−	UUCUGGACCCAGGAUCUUAG	20	2512
  CCR5-3107	−	CACUAUGCCAGAUACAUAGG	20	2513
  CCR5-3108	+	UAGGCUUCCCUCUUGUCUGG	20	2514
  CCR5-3109	−	UGCAUGACAUUCAUCUGUGG	20	2515
  CCR5-3110	−	GUCAUAUCAAGCUCUCUUGG	20	2516
  CCR5-3111	+	GACCAUAUACUUAUGUCAUG	20	2517
  CCR5-3112	−	CAAACCAGUAAGCAUUUAUG	20	2518
  CCR5-3113	−	GAAUGCAUGACAUUCAUCUG	20	2519
  CCR5-3114	−	CUGGACCCAGGAUCUUAGUG	20	2520
  CCR5-3115	−	CAAACUUCACAGAAAAUGUG	20	2521
  CCR5-3116	−	UGUAUGACAACUCUUAAUUG	20	2522
  CCR5-3117	+	GAAGCCCAGAGGGCAUCUUG	20	2523
  CCR5-3118	+	GACUUAGACACAACUUCUUG	20	2524
  CCR5-3119	+	GCACGUAAUUUUGCUGUUUG	20	2525
  CCR5-3120	−	AAAUGUGAGGAUUUUACAAU	20	2526
  CCR5-3121	−	UCACACUAUGCCAGAUACAU	20	2527
  CCR5-3122	+	UCCUGGGUCCAGAAAAAGAU	20	2528
  CCR5-3123	−	GUCUAAAGAGUUUUAACUAU	20	2529
  CCR5-3124	−	CAGCUGAACUUAAAUAGACU	20	2530
  CCR5-3125	+	AACUCCCUGCACCUUAGACU	20	2531
  CCR5-3126	−	CCUCUGGGCUUCCGUGACCU	20	2532
  CCR5-3127	−	UGUCAUCUAUGACCUUCCCU	20	2533
  CCR5-3128	+	AAAAUCCCCACUAAGAUCCU	20	2534
  CCR5-3129	+	CCAGAGGGCAUCUUGUGGCU	20	2535
  CCR5-3130	−	CGAGCCACAAGAUGCCCUCU	20	2536
  CCR5-3131	−	ACAGUCAUAUCAAGCUCUCU	20	2537
  CCR5-3132	+	AGAUGAAUGUCAUGCAUUCU	20	2538
  CCR5-3133	−	UUUUUUAUUAUAUUAUUUCU	20	2539
  CCR5-3134	−	AAUUAAAAAUGAGCUUUUCU	20	2540
  CCR5-3135	−	UCUGGACCCAGGAUCUUAGU	20	2541
  CCR5-3136	−	UAUCAAGCUCUCUUGGCGGU	20	2542
  CCR5-3137	+	ACUUAGACACAACUUCUUGU	20	2543
  CCR5-3138	+	UAUUAGAGGAAAAUUUUAUU	20	2544
  CCR5-3139	−	GGCUCUAGAAUAAAAAGCUU	20	2545
  CCR5-3140	−	UUUUUAUUAUAUUAUUUCUU	20	2546
  CCR5-3141	+	GGGCACGUAAUUUUGCUGUU	20	2547
  CCR5-3142	+	GGCACGUAAUUUUGCUGUUU	20	2548
  CCR5-3143	+	UAUUAAUUUUGACCAUUUUU	20	2549

• Table 6A provides exemplary targeting domains for knocking down the CCR5 gene selected according to the first tier parameters. The targeting domains bind within 500 bp (e.g., upstream or downstream) of a transcription start site (TSS), have a high level of orthogonality and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 6A
  1st Tier

  gRNA	DNA		Target Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-3144	+	AAGUGUAUUGAAGGCGAA	18	2550
  CCR5-3145	+	UAAGUGUAUUGAAGGCGAA	19	2551
  CCR5-3146	+	UUAAGUGUAUUGAAGGCGAA	20	2552
  CCR5-3147	+	AUUAAGUGUAUUGAAGGCGAA	21	2553
  CCR5-3148	+	CAUUAAGUGUAUUGAAGGCGAA	22	2554
  CCR5-3149	+	UCAUUAAGUGUAUUGAAGGCGAA	23	2555
  CCR5-3150	+	AUCAUUAAGUGUAUUGAAGGCGAA	24	2556
  CCR5-3151	+	UUCUCUGCUCAUCCCACUACA	21	2557
  CCR5-3152	+	GUUCUCUGCUCAUCCCACUACA	22	2558
  CCR5-3153	+	UGUUCUCUGCUCAUCCCACUACA	23	2559
  CCR5-3154	+	UUGUUCUCUGCUCAUCCCACUACA	24	2560
  CCR5-3155	+	AUUUACGGGCUUUUCUCA	18	2561
  CCR5-3156	+	UAUUUACGGGCUUUUCUCA	19	2562
  CCR5-3157	+	UUAUUUACGGGCUUUUCUCA	20	2563
  CCR5-3158	+	UUUAUUUACGGGCUUUUCUCA	21	2564
  CCR5-3159	+	GUUUAUUUACGGGCUUUUCUCA	22	2565
  CCR5-3160	+	GGUUUAUUUACGGGCUUUUCUCA	23	2566
  CCR5-3161	+	AGGUUUAUUUACGGGCUUUUCUCA	24	2567
  CCR5-3162	+	GGGAGAGUUUCUUGUAGGGGA	21	2568
  CCR5-3163	+	GGGGAGAGUUUCUUGUAGGGGA	22	2569
  CCR5-3164	+	CGGGGAGAGUUUCUUGUAGGGGA	23	2570
  CCR5-3165	+	CCGGGGAGAGUUUCUUGUAGGGGA	24	2571
  CCR5-3166	+	UUCAGAAGGCAUCUCACUGGA	21	2572
  CCR5-3167	+	AUUCAGAAGGCAUCUCACUGGA	22	2573
  CCR5-3168	+	UAUUCAGAAGGCAUCUCACUGGA	23	2574
  CCR5-3169	+	AUAUUCAGAAGGCAUCUCACUGGA	24	2575
  CCR5-3170	+	UGAGCUUAAAAUAAGCUA	18	2576
  CCR5-3171	+	UUGAGCUUAAAAUAAGCUA	19	2577
  CCR5-3172	+	GUUGAGCUUAAAAUAAGCUA	20	2578
  CCR5-3173	+	GAAAUGCUGUUUCUUUUGAAG	21	2579
  CCR5-3174	+	GGAAAUGCUGUUUCUUUUGAAG	22	2580
  CCR5-3175	+	AGGAAAUGCUGUUUCUUUUGAAG	23	2581
  CCR5-3176	+	UAGGAAAUGCUGUUUCUUUUGAAG	24	2582
  CCR5-3177	+	AAACCAACUUUAAAUGUAGAG	21	2583
  CCR5-3178	+	UAAACCAACUUUAAAUGUAGAG	22	2584
  CCR5-3179	+	UUAAACCAACUUUAAAUGUAGAG	23	2585
  CCR5-3180	+	CUUAAACCAACUUUAAAUGUAGAG	24	2586
  CCR5-3181	+	GCUGUUUCUUUUGAAGGAGGG	21	2587
  CCR5-3182	+	UGCUGUUUCUUUUGAAGGAGGG	22	2588
  CCR5-3183	+	AUGCUGUUUCUUUUGAAGGAGGG	23	2589
  CCR5-3184	+	AAUGCUGUUUCUUUUGAAGGAGGG	24	2590
  CCR5-3185	+	GCUGAGAGGUUACUUACCGGG	21	2591
  CCR5-3186	+	AGCUGAGAGGUUACUUACCGGG	22	2592
  CCR5-3187	+	CAGCUGAGAGGUUACUUACCGGG	23	2593
  CCR5-3188	+	GCAGCUGAGAGGUUACUUACCGGG	24	2594
  CCR5-3189	+	CAAAUCUUUCUUUUGAGAGGU	21	2595
  CCR5-3190	+	GCAAAUCUUUCUUUUGAGAGGU	22	2596
  CCR5-3191	+	UGCAAAUCUUUCUUUUGAGAGGU	23	2597
  CCR5-3192	+	CUGCAAAUCUUUCUUUUGAGAGGU	24	2598
  CCR5-3193	−	AGGAAAGGGUCACAGUUUGGA	21	2599
  CCR5-3194	−	AAGGAAAGGGUCACAGUUUGGA	22	2600
  CCR5-3195	−	UAAGGAAAGGGUCACAGUUUGGA	23	2601
  CCR5-3196	−	AUAAGGAAAGGGUCACAGUUUGGA	24	2602
  CCR5-3197	−	ACACAGGGUUAAUGUGAAGUC	21	2603
  CCR5-3198	−	GACACAGGGUUAAUGUGAAGUC	22	2604
  CCR5-3199	−	GGACACAGGGUUAAUGUGAAGUC	23	2605
  CCR5-3200	−	GGGACACAGGGUUAAUGUGAAGUC	24	2606
  CCR5-3201	−	GCCUGUUAGUUAGCUUCUGAG	21	2607
  CCR5-3202	−	GGCCUGUUAGUUAGCUUCUGAG	22	2608
  CCR5-3203	−	UGGCCUGUUAGUUAGCUUCUGAG	23	2609
  CCR5-3204	−	UUGGCCUGUUAGUUAGCUUCUGAG	24	2610
  CCR5-3205	−	AUGUGGGCUUUUGACUAG	18	2611
  CCR5-3206	−	AAUGUGGGCUUUUGACUAG	19	2612
  CCR5-3207	−	AAAUGUGGGCUUUUGACUAG	20	2613
  CCR5-3208	−	AAAAUGUGGGCUUUUGACUAG	21	2614
  CCR5-3209	−	UAAAAUGUGGGCUUUUGACUAG	22	2615
  CCR5-3210	−	CUAAAAUGUGGGCUUUUGACUAG	23	2616
  CCR5-3211	−	ACUAAAAUGUGGGCUUUUGACUAG	24	2617
  CCR5-3212	−	UUUCUAACAGAUUCUGUGUAG	21	2618
  CCR5-3213	−	UUUUCUAACAGAUUCUGUGUAG	22	2619
  CCR5-3214	−	AUUUUCUAACAGAUUCUGUGUAG	23	2620
  CCR5-3215	−	UAUUUUCUAACAGAUUCUGUGUAG	24	2621
  CCR5-3216	−	GGGUGGGAUAGGGGAUACGGG	21	2622
  CCR5-3217	−	GGGGUGGGAUAGGGGAUACGGG	22	2623
  CCR5-3218	−	UGGGGUGGGAUAGGGGAUACGGG	23	2624
  CCR5-3219	−	UUGGGGUGGGAUAGGGGAUACGGG	24	2625
  CCR5-3220	−	AGCAACUCUUAAGAUAAU	18	2626
  CCR5-3221	−	UAGCAACUCUUAAGAUAAU	19	2627
  CCR5-3222	−	AUAGCAACUCUUAAGAUAAU	20	2628
  CCR5-3223	−	AAUAGCAACUCUUAAGAUAAU	21	2629
  CCR5-3224	−	UAAUAGCAACUCUUAAGAUAAU	22	2630
  CCR5-3225	−	UUAAUAGCAACUCUUAAGAUAAU	23	2631
  CCR5-3226	−	AUUAAUAGCAACUCUUAAGAUAAU	24	2632
  CCR5-3227	−	GGUGAGCAUCUGUGUGGGGGU	21	2633
  CCR5-3228	−	UGGUGAGCAUCUGUGUGGGGGU	22	2634
  CCR5-3229	−	GUGGUGAGCAUCUGUGUGGGGGU	23	2635
  CCR5-3230	−	GGUGGUGAGCAUCUGUGUGGGGGU	24	2636
  CCR5-3231	−	UUGGGUGGUGAGCAUCUGUGU	21	2637
  CCR5-3232	−	AUUGGGUGGUGAGCAUCUGUGU	22	2638
  CCR5-3233	−	UAUUGGGUGGUGAGCAUCUGUGU	23	2639
  CCR5-3234	−	AUAUUGGGUGGUGAGCAUCUGUGU	24	2640
  CCR5-3235	−	UCAAAGAUACAAAACAUGAUU	21	2641
  CCR5-3236	−	AUCAAAGAUACAAAACAUGAUU	22	2642
  CCR5-3237	−	CAUCAAAGAUACAAAACAUGAUU	23	2643
  CCR5-3238	−	ACAUCAAAGAUACAAAACAUGAUU	24	2644
  CCR5-3239	−	CCCUCUCCAGUGAGAUGCCUU	21	2645
  CCR5-3240	−	ACCCUCUCCAGUGAGAUGCCUU	22	2646
  CCR5-3241	−	AACCCUCUCCAGUGAGAUGCCUU	23	2647
  CCR5-3242	−	AAACCCUCUCCAGUGAGAUGCCUU	24	2648

• Table 6B provides exemplary targeting domains for knocking down the CCR5 gene selected according to the second tier parameters. The targeting domains bind within 500 bp (e.g., upstream or downstream) of a transcription start site (TSS) and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 6B
  2nd Tier

  gRNA	DNA		Target Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-3243	+	UCUGCUCAUCCCACUACA	18	2649
  CCR5-3244	+	CUCUGCUCAUCCCACUACA	19	2650
  CCR5-3245	+	UCUCUGCUCAUCCCACUACA	20	2651
  CCR5-3246	+	AGAGUUUCUUGUAGGGGA	18	2652
  CCR5-3247	+	GAGAGUUUCUUGUAGGGGA	19	2653
  CCR5-3248	+	GGAGAGUUUCUUGUAGGGGA	20	2654
  CCR5-3249	+	AGAAGGCAUCUCACUGGA	18	2655
  CCR5-3250	+	CAGAAGGCAUCUCACUGGA	19	2656
  CCR5-3251	+	UCAGAAGGCAUCUCACUGGA	20	2657
  CCR5-3252	+	UAGAAAAUAUAAAGAAUA	18	2658
  CCR5-3253	+	UUAGAAAAUAUAAAGAAUA	19	2659
  CCR5-3254	+	GUUAGAAAAUAUAAAGAAUA	20	2660
  CCR5-3255	+	UGUUAGAAAAUAUAAAGAAUA	21	2661
  CCR5-3256	+	CUGUUAGAAAAUAUAAAGAAUA	22	2662
  CCR5-3257	+	UCUGUUAGAAAAUAUAAAGAAUA	23	2663
  CCR5-3258	+	AUCUGUUAGAAAAUAUAAAGAAUA	24	2664
  CCR5-3259	+	AAUCUGUUAGAAAAUAUA	18	2665
  CCR5-3260	+	GAAUCUGUUAGAAAAUAUA	19	2666
  CCR5-3261	+	AGAAUCUGUUAGAAAAUAUA	20	2667
  CCR5-3262	+	CAGAAUCUGUUAGAAAAUAUA	21	2668
  CCR5-3263	+	ACAGAAUCUGUUAGAAAAUAUA	22	2669
  CCR5-3264	+	CACAGAAUCUGUUAGAAAAUAUA	23	2670
  CCR5-3265	+	ACACAGAAUCUGUUAGAAAAUAUA	24	2671
  CCR5-3266	+	AGUUGAGCUUAAAAUAAGCUA	21	2672
  CCR5-3267	+	AAGUUGAGCUUAAAAUAAGCUA	22	2673
  CCR5-3268	+	UAAGUUGAGCUUAAAAUAAGCUA	23	2674
  CCR5-3269	+	UUAAGUUGAGCUUAAAAUAAGCUA	24	2675
  CCR5-3270	+	AUGCUGUUUCUUUUGAAG	18	2676
  CCR5-3271	+	AAUGCUGUUUCUUUUGAAG	19	2677
  CCR5-3272	+	AAAUGCUGUUUCUUUUGAAG	20	2678
  CCR5-3273	+	CCAACUUUAAAUGUAGAG	18	2679
  CCR5-3274	+	ACCAACUUUAAAUGUAGAG	19	2680
  CCR5-2944	+	AACCAACUUUAAAUGUAGAG	20	2681
  CCR5-3275	+	GUUUCUUUUGAAGGAGGG	18	2682
  CCR5-3276	+	UGUUUCUUUUGAAGGAGGG	19	2683
  CCR5-2954	+	CUGUUUCUUUUGAAGGAGGG	20	2684
  CCR5-3277	+	GAGAGGUUACUUACCGGG	18	2685
  CCR5-3278	+	UGAGAGGUUACUUACCGGG	19	2686
  CCR5-3279	+	CUGAGAGGUUACUUACCGGG	20	2687
  CCR5-3280	+	GUUUGCCAAAUGUCUUCU	18	2688
  CCR5-3281	+	UGUUUGCCAAAUGUCUUCU	19	2689
  CCR5-3282	+	GUGUUUGCCAAAUGUCUUCU	20	2690
  CCR5-3283	+	GGUGUUUGCCAAAUGUCUUCU	21	2691
  CCR5-3284	+	UGGUGUUUGCCAAAUGUCUUCU	22	2692
  CCR5-3285	+	UUGGUGUUUGCCAAAUGUCUUCU	23	2693
  CCR5-3286	+	CUUGGUGUUUGCCAAAUGUCUUCU	24	2694
  CCR5-3287	+	AUCUUUCUUUUGAGAGGU	18	2695
  CCR5-3288	+	AAUCUUUCUUUUGAGAGGU	19	2696
  CCR5-3289	+	AAAUCUUUCUUUUGAGAGGU	20	2697
  CCR5-3290	+	GAAAAUUCUGAUUAUCUU	18	2698
  CCR5-3291	+	AGAAAAUUCUGAUUAUCUU	19	2699
  CCR5-3292	+	AAGAAAAUUCUGAUUAUCUU	20	2700
  CCR5-3293	+	UAAGAAAAUUCUGAUUAUCUU	21	2701
  CCR5-3294	+	UUAAGAAAAUUCUGAUUAUCUU	22	2702
  CCR5-3295	+	GUUAAGAAAAUUCUGAUUAUCUU	23	2703
  CCR5-3296	+	GGUUAAGAAAAUUCUGAUUAUCUU	24	2704
  CCR5-3297	−	GUGGAGAAAAAGGGGACA	18	2705
  CCR5-3298	−	AGUGGAGAAAAAGGGGACA	19	2706
  CCR5-3299	−	GAGUGGAGAAAAAGGGGACA	20	2707
  CCR5-3300	−	AGAGUGGAGAAAAAGGGGACA	21	2708
  CCR5-3301	−	GAGAGUGGAGAAAAAGGGGACA	22	2709
  CCR5-3302	−	GGAGAGUGGAGAAAAAGGGGACA	23	2710
  CCR5-3303	−	GGGAGAGUGGAGAAAAAGGGGACA	24	2711
  CCR5-3304	−	UAAUCUUUAAGAUAAGGA	18	2712
  CCR5-3305	−	AUAAUCUUUAAGAUAAGGA	19	2713
  CCR5-3306	−	UAUAAUCUUUAAGAUAAGGA	20	2714
  CCR5-3307	−	AUAUAAUCUUUAAGAUAAGGA	21	2715
  CCR5-3308	−	AAUAUAAUCUUUAAGAUAAGGA	22	2716
  CCR5-3309	−	AAAUAUAAUCUUUAAGAUAAGGA	23	2717
  CCR5-3310	−	AAAAUAUAAUCUUUAAGAUAAGGA	24	2718
  CCR5-3311	−	AAAGGGUCACAGUUUGGA	18	2719
  CCR5-3312	−	GAAAGGGUCACAGUUUGGA	19	2720
  CCR5-3313	−	GGAAAGGGUCACAGUUUGGA	20	2721
  CCR5-3314	−	UUACAGAGAACAAUAAUA	18	2722
  CCR5-3315	−	UUUACAGAGAACAAUAAUA	19	2723
  CCR5-3316	−	GUUUACAGAGAACAAUAAUA	20	2724
  CCR5-3317	−	GGGGGUUGGGGUGGGAUA	18	2725
  CCR5-3318	−	UGGGGGUUGGGGUGGGAUA	19	2726
  CCR5-2924	−	GUGGGGGUUGGGGUGGGAUA	20	2727
  CCR5-3319	−	UGUGGGGGUUGGGGUGGGAUA	21	2728
  CCR5-3320	−	GUGUGGGGGUUGGGGUGGGAUA	22	2729
  CCR5-3321	−	UGUGUGGGGGUUGGGGUGGGAUA	23	2730
  CCR5-3322	−	CUGUGUGGGGGUUGGGGUGGGAUA	24	2731
  CCR5-3323	−	CAGGGUUAAUGUGAAGUC	18	2732
  CCR5-3324	−	ACAGGGUUAAUGUGAAGUC	19	2733
  CCR5-3325	−	CACAGGGUUAAUGUGAAGUC	20	2734
  CCR5-3326	−	GUACAAAUCAUUUGCUUC	18	2735
  CCR5-3327	−	UGUACAAAUCAUUUGCUUC	19	2736
  CCR5-3328	−	UUGUACAAAUCAUUUGCUUC	20	2737
  CCR5-3329	−	CUUGUACAAAUCAUUUGCUUC	21	2738
  CCR5-3330	−	UCUUGUACAAAUCAUUUGCUUC	22	2739
  CCR5-3331	−	AUCUUGUACAAAUCAUUUGCUUC	23	2740
  CCR5-3332	−	GAUCUUGUACAAAUCAUUUGCUUC	24	2741
  CCR5-3333	−	AGAAAGAUUUGCAGAGAG	18	2742
  CCR5-3334	−	AAGAAAGAUUUGCAGAGAG	19	2743
  CCR5-3335	−	AAAGAAAGAUUUGCAGAGAG	20	2744
  CCR5-3336	−	AAAAGAAAGAUUUGCAGAGAG	21	2745
  CCR5-3337	−	CAAAAGAAAGAUUUGCAGAGAG	22	2746
  CCR5-3338	−	UCAAAAGAAAGAUUUGCAGAGAG	23	2747
  CCR5-3339	−	CUCAAAAGAAAGAUUUGCAGAGAG	24	2748
  CCR5-3340	−	UGUUAGUUAGCUUCUGAG	18	2749
  CCR5-3341	−	CUGUUAGUUAGCUUCUGAG	19	2750
  CCR5-3342	−	CCUGUUAGUUAGCUUCUGAG	20	2751
  CCR5-3343	−	CUAACAGAUUCUGUGUAG	18	2752
  CCR5-3344	−	UCUAACAGAUUCUGUGUAG	19	2753
  CCR5-2950	−	UUCUAACAGAUUCUGUGUAG	20	2754
  CCR5-3345	−	UGGGAUAGGGGAUACGGG	18	2755
  CCR5-3346	−	GUGGGAUAGGGGAUACGGG	19	2756
  CCR5-3347	−	GGUGGGAUAGGGGAUACGGG	20	2757
  CCR5-3348	−	UCUGUGUGGGGGUUGGGG	18	2758
  CCR5-3349	−	AUCUGUGUGGGGGUUGGGG	19	2759
  CCR5-2955	−	CAUCUGUGUGGGGGUUGGGG	20	2760
  CCR5-3350	−	GCAUCUGUGUGGGGGUUGGGG	21	2761
  CCR5-3351	−	AGCAUCUGUGUGGGGGUUGGGG	22	2762
  CCR5-3352	−	GAGCAUCUGUGUGGGGGUUGGGG	23	2763
  CCR5-3353	−	UGAGCAUCUGUGUGGGGGUUGGGG	24	2764
  CCR5-3354	−	GAGCAUCUGUGUGGGGGU	18	2765
  CCR5-3355	−	UGAGCAUCUGUGUGGGGGU	19	2766
  CCR5-2970	−	GUGAGCAUCUGUGUGGGGGU	20	2767
  CCR5-3356	−	GGUGGUGAGCAUCUGUGU	18	2768
  CCR5-3357	−	GGGUGGUGAGCAUCUGUGU	19	2769
  CCR5-2973	−	UGGGUGGUGAGCAUCUGUGU	20	2770
  CCR5-3358	−	AAGAUACAAAACAUGAUU	18	2771
  CCR5-3359	−	AAAGAUACAAAACAUGAUU	19	2772
  CCR5-3360	−	CAAAGAUACAAAACAUGAUU	20	2773
  CCR5-3361	−	UCUCCAGUGAGAUGCCUU	18	2774
  CCR5-3362	−	CUCUCCAGUGAGAUGCCUU	19	2775
  CCR5-3363	−	CCUCUCCAGUGAGAUGCCUU	20	2776
  CCR5-3364	−	AAGGAAAGGGUCACAGUU	18	2777
  CCR5-3365	−	UAAGGAAAGGGUCACAGUU	19	2778
  CCR5-2977	−	AUAAGGAAAGGGUCACAGUU	20	2779
  CCR5-3366	−	GAUAAGGAAAGGGUCACAGUU	21	2780
  CCR5-3367	−	AGAUAAGGAAAGGGUCACAGUU	22	2781
  CCR5-3368	−	AAGAUAAGGAAAGGGUCACAGUU	23	2782
  CCR5-3369	−	UAAGAUAAGGAAAGGGUCACAGUU	24	2783

• Table 6C provides exemplary targeting domains for knocking down the CCR5 gene selected according to the third tier parameters. The targeting domains bind within 500 bp (e.g., upstream or downstream) of a transcription start site (TSS) and PAM is NNGRRV. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 6C
  3rd Tier

  gRNA	DNA		Target Site
  Name	Strand	Targeting Domain	Length	SEQ ID NO
  CCR5-4045	+	GGGCAACAAAAUAGUGAA	18	3483
  CCR5-4046	+	AGGGCAACAAAAUAGUGAA	19	3484
  CCR5-4047	+	AAGGGCAACAAAAUAGUGAA	20	3485
  CCR5-4048	+	GAAGGGCAACAAAAUAGUGAA	21	3486
  CCR5-4049	+	UGAAGGGCAACAAAAUAGUGAA	22	3487
  CCR5-4050	+	UUGAAGGGCAACAAAAUAGUGAA	23	3488
  CCR5-4051	+	UUUGAAGGGCAACAAAAUAGUGAA	24	3489
  CCR5-4052	+	UUUUAAUUUUGAACCAUA	18	3490
  CCR5-4053	+	UUUUUAAUUUUGAACCAUA	19	3491
  CCR5-4054	+	AUUUUUAAUUUUGAACCAUA	20	3492
  CCR5-4055	+	CAUUUUUAAUUUUGAACCAUA	21	3493
  CCR5-4056	+	UCAUUUUUAAUUUUGAACCAUA	22	3494
  CCR5-4057	+	CUCAUUUUUAAUUUUGAACCAUA	23	3495
  CCR5-4058	+	GCUCAUUUUUAAUUUUGAACCAUA	24	3496
  CCR5-4059	+	AAAAUCCCCACUAAGAUC	18	3497
  CCR5-4060	+	GAAAAUCCCCACUAAGAUC	19	3498
  CCR5-4061	+	UGAAAAUCCCCACUAAGAUC	20	3499
  CCR5-4062	+	GUGAAAAUCCCCACUAAGAUC	21	3500
  CCR5-4063	+	AGUGAAAAUCCCCACUAAGAUC	22	3501
  CCR5-4064	+	GAGUGAAAAUCCCCACUAAGAUC	23	3502
  CCR5-4065	+	AGAGUGAAAAUCCCCACUAAGAUC	24	3503
  CCR5-4066	+	CUUCAGAUAGAUUAUAUC	18	3504
  CCR5-4067	+	GCUUCAGAUAGAUUAUAUC	19	3505
  CCR5-3092	+	AGCUUCAGAUAGAUUAUAUC	20	3506
  CCR5-4068	+	UAGCUUCAGAUAGAUUAUAUC	21	3507
  CCR5-4069	+	AUAGCUUCAGAUAGAUUAUAUC	22	3508
  CCR5-4070	+	CAUAGCUUCAGAUAGAUUAUAUC	23	3509
  CCR5-4071	+	UCAUAGCUUCAGAUAGAUUAUAUC	24	3510
  CCR5-4072	+	GAGGGCAUCUUGUGGCUC	18	3511
  CCR5-4073	+	AGAGGGCAUCUUGUGGCUC	19	3512
  CCR5-3095	+	CAGAGGGCAUCUUGUGGCUC	20	3513
  CCR5-4074	+	CCAGAGGGCAUCUUGUGGCUC	21	3514
  CCR5-4075	+	CCCAGAGGGCAUCUUGUGGCUC	22	3515
  CCR5-4076	+	GCCCAGAGGGCAUCUUGUGGCUC	23	3516
  CCR5-4077	+	AGCCCAGAGGGCAUCUUGUGGCUC	24	3517
  CCR5-4078	+	UUUCGUCUGCCACCACAG	18	3518
  CCR5-4079	+	GUUUCGUCUGCCACCACAG	19	3519
  CCR5-4080	+	UGUUUCGUCUGCCACCACAG	20	3520
  CCR5-4081	+	AUGUUUCGUCUGCCACCACAG	21	3521
  CCR5-4082	+	AAUGUUUCGUCUGCCACCACAG	22	3522
  CCR5-4083	+	AAAUGUUUCGUCUGCCACCACAG	23	3523
  CCR5-4084	+	AAAAUGUUUCGUCUGCCACCACAG	24	3524
  CCR5-4085	+	UAGAUUAUAUCUGGAGUG	18	3525
  CCR5-4086	+	AUAGAUUAUAUCUGGAGUG	19	3526
  CCR5-4087	+	GAUAGAUUAUAUCUGGAGUG	20	3527
  CCR5-4088	+	AGAUAGAUUAUAUCUGGAGUG	21	3528
  CCR5-4089	+	CAGAUAGAUUAUAUCUGGAGUG	22	3529
  CCR5-4090	+	UCAGAUAGAUUAUAUCUGGAGUG	23	3530
  CCR5-4091	+	UUCAGAUAGAUUAUAUCUGGAGUG	24	3531
  CCR5-4092	+	UUUCUCUUAUUAAACCCU	18	3532
  CCR5-4093	+	UUUUCUCUUAUUAAACCCU	19	3533
  CCR5-4094	+	AUUUUCUCUUAUUAAACCCU	20	3534
  CCR5-4095	+	AAUUUUCUCUUAUUAAACCCU	21	3535
  CCR5-4096	+	GAAUUUUCUCUUAUUAAACCCU	22	3536
  CCR5-4097	+	AGAAUUUUCUCUUAUUAAACCCU	23	3537
  CCR5-4098	+	GAGAAUUUUCUCUUAUUAAACCCU	24	3538
  CCR5-4099	+	AGUUCAGCUGCUCUAGCU	18	3539
  CCR5-4100	+	AAGUUCAGCUGCUCUAGCU	19	3540
  CCR5-4101	+	UAAGUUCAGCUGCUCUAGCU	20	3541
  CCR5-4102	+	UUAAGUUCAGCUGCUCUAGCU	21	3542
  CCR5-4103	+	UUUAAGUUCAGCUGCUCUAGCU	22	3543
  CCR5-4104	+	AUUUAAGUUCAGCUGCUCUAGCU	23	3544
  CCR5-4105	+	UAUUUAAGUUCAGCUGCUCUAGCU	24	3545
  CCR5-4106	+	CUAUGUAUCUGGCAUAGU	18	3546
  CCR5-4107	+	CCUAUGUAUCUGGCAUAGU	19	3547
  CCR5-4108	+	ACCUAUGUAUCUGGCAUAGU	20	3548
  CCR5-4109	+	CACCUAUGUAUCUGGCAUAGU	21	3549
  CCR5-4110	+	CCACCUAUGUAUCUGGCAUAGU	22	3550
  CCR5-4111	+	GCCACCUAUGUAUCUGGCAUAGU	23	3551
  CCR5-4112	+	UGCCACCUAUGUAUCUGGCAUAGU	24	3552
  CCR5-4113	+	UUCUGAGUUGCCACAAUU	18	3553
  CCR5-4114	+	UUUCUGAGUUGCCACAAUU	19	3554
  CCR5-4115	+	GUUUCUGAGUUGCCACAAUU	20	3555
  CCR5-4116	+	AGUUUCUGAGUUGCCACAAUU	21	3556
  CCR5-4117	+	UAGUUUCUGAGUUGCCACAAUU	22	3557
  CCR5-4118	+	GUAGUUUCUGAGUUGCCACAAUU	23	3558
  CCR5-4119	+	UGUAGUUUCUGAGUUGCCACAAUU	24	3559
  CCR5-4120	+	AGAUGAAUGUCAUGCAUU	18	3560
  CCR5-4121	+	CAGAUGAAUGUCAUGCAUU	19	3561
  CCR5-4122	+	ACAGAUGAAUGUCAUGCAUU	20	3562
  CCR5-4123	+	CACAGAUGAAUGUCAUGCAUU	21	3563
  CCR5-4124	+	CCACAGAUGAAUGUCAUGCAUU	22	3564
  CCR5-4125	+	ACCACAGAUGAAUGUCAUGCAUU	23	3565
  CCR5-4126	+	CACCACAGAUGAAUGUCAUGCAUU	24	3566
  CCR5-4127	+	GCACGUAAUUUUGCUGUU	18	3567
  CCR5-4128	+	GGCACGUAAUUUUGCUGUU	19	3568
  CCR5-3141	+	GGGCACGUAAUUUUGCUGUU	20	3569
  CCR5-4129	+	GGGGCACGUAAUUUUGCUGUU	21	3570
  CCR5-4130	+	GGGGGCACGUAAUUUUGCUGUU	22	3571
  CCR5-4131	+	UGGGGGCACGUAAUUUUGCUGUU	23	3572
  CCR5-4132	+	UUGGGGGCACGUAAUUUUGCUGUU	24	3573
  CCR5-4133	+	AGUUUGUGUUUGUAGUUU	18	3574
  CCR5-4134	+	AAGUUUGUGUUUGUAGUUU	19	3575
  CCR5-4135	+	GAAGUUUGUGUUUGUAGUUU	20	3576
  CCR5-4136	+	UGAAGUUUGUGUUUGUAGUUU	21	3577
  CCR5-4137	+	GUGAAGUUUGUGUUUGUAGUUU	22	3578
  CCR5-4138	+	UGUGAAGUUUGUGUUUGUAGUUU	23	3579
  CCR5-4139	+	CUGUGAAGUUUGUGUUUGUAGUUU	24	3580
  CCR5-4140	−	UGCCUAGUCUAAGGUGCA	18	3581
  CCR5-4141	−	CUGCCUAGUCUAAGGUGCA	19	3582
  CCR5-3067	−	GCUGCCUAGUCUAAGGUGCA	20	3583
  CCR5-4142	−	AGCUGCCUAGUCUAAGGUGCA	21	3584
  CCR5-4143	−	CAGCUGCCUAGUCUAAGGUGCA	22	3585
  CCR5-4144	−	UCAGCUGCCUAGUCUAAGGUGCA	23	3586
  CCR5-4145	−	CUCAGCUGCCUAGUCUAAGGUGCA	24	3587
  CCR5-4146	−	CAGGGAGUUUGAGACUCA	18	3588
  CCR5-4147	−	GCAGGGAGUUUGAGACUCA	19	3589
  CCR5-4148	−	UGCAGGGAGUUUGAGACUCA	20	3590
  CCR5-4149	−	GUGCAGGGAGUUUGAGACUCA	21	3591
  CCR5-4150	−	GGUGCAGGGAGUUUGAGACUCA	22	3592
  CCR5-4151	−	AGGUGCAGGGAGUUUGAGACUCA	23	3593
  CCR5-4152	−	AAGGUGCAGGGAGUUUGAGACUCA	24	3594
  CCR5-4153	−	CCCAUCUUUUUCUGGACC	18	3595
  CCR5-4154	−	UCCCAUCUUUUUCUGGACC	19	3596
  CCR5-4155	−	UUCCCAUCUUUUUCUGGACC	20	3597
  CCR5-4156	−	UUUCCCAUCUUUUUCUGGACC	21	3598
  CCR5-4157	−	GUUUCCCAUCUUUUUCUGGACC	22	3599
  CCR5-4158	−	GGUUUCCCAUCUUUUUCUGGACC	23	3600
  CCR5-4159	−	AGGUUUCCCAUCUUUUUCUGGACC	24	3601
  CCR5-4160	−	UUAUAAGACUAAACUACC	18	3602
  CCR5-4161	−	GUUAUAAGACUAAACUACC	19	3603
  CCR5-4162	−	GGUUAUAAGACUAAACUACC	20	3604
  CCR5-4163	−	UGGUUAUAAGACUAAACUACC	21	3605
  CCR5-4164	−	CUGGUUAUAAGACUAAACUACC	22	3606
  CCR5-4165	−	GCUGGUUAUAAGACUAAACUACC	23	3607
  CCR5-4166	−	AGCUGGUUAUAAGACUAAACUACC	24	3608
  CCR5-4167	−	AGUUUUAACUAUGGGCUC	18	3609
  CCR5-4168	−	GAGUUUUAACUAUGGGCUC	19	3610
  CCR5-4169	−	AGAGUUUUAACUAUGGGCUC	20	3611
  CCR5-4170	−	AAGAGUUUUAACUAUGGGCUC	21	3612
  CCR5-4171	−	AAAGAGUUUUAACUAUGGGCUC	22	3613
  CCR5-4172	−	UAAAGAGUUUUAACUAUGGGCUC	23	3614
  CCR5-4173	−	CUAAAGAGUUUUAACUAUGGGCUC	24	3615
  CCR5-4174	−	CUUCCGUGACCUUGGCUC	18	3616
  CCR5-4175	−	GCUUCCGUGACCUUGGCUC	19	3617
  CCR5-4176	−	GGCUUCCGUGACCUUGGCUC	20	3618
  CCR5-4177	−	GGGCUUCCGUGACCUUGGCUC	21	3619
  CCR5-4178	−	UGGGCUUCCGUGACCUUGGCUC	22	3620
  CCR5-4179	−	CUGGGCUUCCGUGACCUUGGCUC	23	3621
  CCR5-4180	−	UCUGGGCUUCCGUGACCUUGGCUC	24	3622
  CCR5-4181	−	UUUUUAUUAUAUUAUUUC	18	3623
  CCR5-4182	−	UUUUUUAUUAUAUUAUUUC	19	3624
  CCR5-4183	−	AUUUUUUAUUAUAUUAUUUC	20	3625
  CCR5-4184	−	CAUUUUUUAUUAUAUUAUUUC	21	3626
  CCR5-4185	−	ACAUUUUUUAUUAUAUUAUUUC	22	3627
  CCR5-4186	−	AACAUUUUUUAUUAUAUUAUUUC	23	3628
  CCR5-4187	−	AAACAUUUUUUAUUAUAUUAUUUC	24	3629
  CCR5-4188	−	UGCCAGAUACAUAGGUGG	18	3630
  CCR5-4189	−	AUGCCAGAUACAUAGGUGG	19	3631
  CCR5-4190	−	UAUGCCAGAUACAUAGGUGG	20	3632
  CCR5-4191	−	CUAUGCCAGAUACAUAGGUGG	21	3633
  CCR5-4192	−	ACUAUGCCAGAUACAUAGGUGG	22	3634
  CCR5-4193	−	CACUAUGCCAGAUACAUAGGUGG	23	3635
  CCR5-4194	−	ACACUAUGCCAGAUACAUAGGUGG	24	3636
  CCR5-4195	−	UGGACCCAGGAUCUUAGU	18	3637
  CCR5-4196	−	CUGGACCCAGGAUCUUAGU	19	3638
  CCR5-3135	−	UCUGGACCCAGGAUCUUAGU	20	3639
  CCR5-4197	−	UUCUGGACCCAGGAUCUUAGU	21	3640
  CCR5-4198	−	UUUCUGGACCCAGGAUCUUAGU	22	3641
  CCR5-4199	−	UUUUCUGGACCCAGGAUCUUAGU	23	3642
  CCR5-4200	−	UUUUUCUGGACCCAGGAUCUUAGU	24	3643
  CCR5-4201	−	AAACUUCACAGAAAAUGU	18	3644
  CCR5-4202	−	CAAACUUCACAGAAAAUGU	19	3645
  CCR5-4203	−	ACAAACUUCACAGAAAAUGU	20	3646
  CCR5-4204	−	CACAAACUUCACAGAAAAUGU	21	3647
  CCR5-4205	−	ACACAAACUUCACAGAAAAUGU	22	3648
  CCR5-4206	−	AACACAAACUUCACAGAAAAUGU	23	3649
  CCR5-4207	−	AAACACAAACUUCACAGAAAAUGU	24	3650

• Table 6D provides exemplary targeting domains for knocking down the CCR5 gene selected according to the fourth tier parameters. Within the additional 500 bp (e.g., upstream or downstream) of a transcription start site (TSS), e.g., extending to 1 kb upstream and downstream of a TSS and PAM is NNGRRT. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 6D
  4th Tier

  gRNA	DNA		Target Site
  Name	Strand	Targeting Domain	Length	SEQ ID NO
  CCR5-3370	+	AAGCCCACAUUUUAGUAA	18	2784
  CCR5-3371	+	AAAGCCCACAUUUUAGUAA	19	2785
  CCR5-3372	+	AAAAGCCCACAUUUUAGUAA	20	2786
  CCR5-3373	+	CAAAAGCCCACAUUUUAGUAA	21	2787
  CCR5-3374	+	UCAAAAGCCCACAUUUUAGUAA	22	2788
  CCR5-3375	+	GUCAAAAGCCCACAUUUUAGUAA	23	2789
  CCR5-3376	+	AGUCAAAAGCCCACAUUUUAGUAA	24	2790
  CCR5-3377	+	UGAAGGCGAAAAGAAUCA	18	2791
  CCR5-3378	+	UUGAAGGCGAAAAGAAUCA	19	2792
  CCR5-3379	+	AUUGAAGGCGAAAAGAAUCA	20	2793
  CCR5-3380	+	UAUUGAAGGCGAAAAGAAUCA	21	2794
  CCR5-3381	+	GUAUUGAAGGCGAAAAGAAUCA	22	2795
  CCR5-3382	+	UGUAUUGAAGGCGAAAAGAAUCA	23	2796
  CCR5-3383	+	GUGUAUUGAAGGCGAAAAGAAUCA	24	2797
  CCR5-3384	+	AUGAUUUGUACAAGAUCA	18	2798
  CCR5-3385	+	AAUGAUUUGUACAAGAUCA	19	2799
  CCR5-3386	+	AAAUGAUUUGUACAAGAUCA	20	2800
  CCR5-3387	+	CAAAUGAUUUGUACAAGAUCA	21	2801
  CCR5-3388	+	GCAAAUGAUUUGUACAAGAUCA	22	2802
  CCR5-3389	+	AGCAAAUGAUUUGUACAAGAUCA	23	2803
  CCR5-3390	+	AAGCAAAUGAUUUGUACAAGAUCA	24	2804
  CCR5-3391	+	UAUUCAGAAGGCAUCUCA	18	2805
  CCR5-3392	+	AUAUUCAGAAGGCAUCUCA	19	2806
  CCR5-3393	+	CAUAUUCAGAAGGCAUCUCA	20	2807
  CCR5-3394	+	ACCAACUUUAAAUGUAGA	18	2808
  CCR5-3395	+	AACCAACUUUAAAUGUAGA	19	2809
  CCR5-2918	+	AAACCAACUUUAAAUGUAGA	20	2810
  CCR5-3396	+	UAAACCAACUUUAAAUGUAGA	21	2811
  CCR5-3397	+	UUAAACCAACUUUAAAUGUAGA	22	2812
  CCR5-3398	+	CUUAAACCAACUUUAAAUGUAGA	23	2813
  CCR5-3399	+	ACUUAAACCAACUUUAAAUGUAGA	24	2814
  CCR5-3400	+	AAAUGCUGUUUCUUUUGA	18	2815
  CCR5-3401	+	GAAAUGCUGUUUCUUUUGA	19	2816
  CCR5-2921	+	GGAAAUGCUGUUUCUUUUGA	20	2817
  CCR5-3402	+	AGGAAAUGCUGUUUCUUUUGA	21	2818
  CCR5-3403	+	UAGGAAAUGCUGUUUCUUUUGA	22	2819
  CCR5-3404	+	GUAGGAAAUGCUGUUUCUUUUGA	23	2820
  CCR5-3405	+	AGUAGGAAAUGCUGUUUCUUUUGA	24	2821
  CCR5-3406	+	AAACCAACUUUAAAUGUA	18	2822
  CCR5-3407	+	UAAACCAACUUUAAAUGUA	19	2823
  CCR5-3408	+	UUAAACCAACUUUAAAUGUA	20	2824
  CCR5-3409	+	CUUAAACCAACUUUAAAUGUA	21	2825
  CCR5-3410	+	ACUUAAACCAACUUUAAAUGUA	22	2826
  CCR5-3411	+	AACUUAAACCAACUUUAAAUGUA	23	2827
  CCR5-3412	+	CAACUUAAACCAACUUUAAAUGUA	24	2828
  CCR5-3413	+	GUUAAAUCAUUAAGUGUA	18	2829
  CCR5-3414	+	AGUUAAAUCAUUAAGUGUA	19	2830
  CCR5-3415	+	GAGUUAAAUCAUUAAGUGUA	20	2831
  CCR5-3416	+	GGAGUUAAAUCAUUAAGUGUA	21	2832
  CCR5-3417	+	UGGAGUUAAAUCAUUAAGUGUA	22	2833
  CCR5-3418	+	GUGGAGUUAAAUCAUUAAGUGUA	23	2834
  CCR5-3419	+	GGUGGAGUUAAAUCAUUAAGUGUA	24	2835
  CCR5-3420	+	CGGGGAGAGUUUCUUGUA	18	2836
  CCR5-3421	+	CCGGGGAGAGUUUCUUGUA	19	2837
  CCR5-2929	+	ACCGGGGAGAGUUUCUUGUA	20	2838
  CCR5-3422	+	UACCGGGGAGAGUUUCUUGUA	21	2839
  CCR5-3423	+	UUACCGGGGAGAGUUUCUUGUA	22	2840
  CCR5-3424	+	CUUACCGGGGAGAGUUUCUUGUA	23	2841
  CCR5-3425	+	ACUUACCGGGGAGAGUUUCUUGUA	24	2842
  CCR5-3426	+	CAGCUGAGAGGUUACUUA	18	2843
  CCR5-3427	+	GCAGCUGAGAGGUUACUUA	19	2844
  CCR5-3428	+	AGCAGCUGAGAGGUUACUUA	20	2845
  CCR5-3429	+	AAGCAGCUGAGAGGUUACUUA	21	2846
  CCR5-3430	+	CAAGCAGCUGAGAGGUUACUUA	22	2847
  CCR5-3431	+	CCAAGCAGCUGAGAGGUUACUUA	23	2848
  CCR5-3432	+	GCCAAGCAGCUGAGAGGUUACUUA	24	2849
  CCR5-3433	+	AUUCAGAAGGCAUCUCAC	18	2850
  CCR5-3434	+	UAUUCAGAAGGCAUCUCAC	19	2851
  CCR5-2932	+	AUAUUCAGAAGGCAUCUCAC	20	2852
  CCR5-3435	+	AGCUGAGAGGUUACUUAC	18	2853
  CCR5-3436	+	CAGCUGAGAGGUUACUUAC	19	2854
  CCR5-2935	+	GCAGCUGAGAGGUUACUUAC	20	2855
  CCR5-3437	+	AGCAGCUGAGAGGUUACUUAC	21	2856
  CCR5-3438	+	AAGCAGCUGAGAGGUUACUUAC	22	2857
  CCR5-3439	+	CAAGCAGCUGAGAGGUUACUUAC	23	2858
  CCR5-3440	+	CCAAGCAGCUGAGAGGUUACUUAC	24	2859
  CCR5-3441	+	GCUGAGAGGUUACUUACC	18	2860
  CCR5-3442	+	AGCUGAGAGGUUACUUACC	19	2861
  CCR5-2937	+	CAGCUGAGAGGUUACUUACC	20	2862
  CCR5-3443	+	GCAGCUGAGAGGUUACUUACC	21	2863
  CCR5-3444	+	AGCAGCUGAGAGGUUACUUACC	22	2864
  CCR5-3445	+	AAGCAGCUGAGAGGUUACUUACC	23	2865
  CCR5-3446	+	CAAGCAGCUGAGAGGUUACUUACC	24	2866
  CCR5-3447	+	UAAAAGAAAUUACUAUCC	18	2867
  CCR5-3448	+	GUAAAAGAAAUUACUAUCC	19	2868
  CCR5-3449	+	AGUAAAAGAAAUUACUAUCC	20	2869
  CCR5-3450	+	UAGUAAAAGAAAUUACUAUCC	21	2870
  CCR5-3451	+	UUAGUAAAAGAAAUUACUAUCC	22	2871
  CCR5-3452	+	UUUAGUAAAAGAAAUUACUAUCC	23	2872
  CCR5-3453	+	UUUUAGUAAAAGAAAUUACUAUCC	24	2873
  CCR5-3454	+	GUUGAGCUUAAAAUAAGC	18	2874
  CCR5-3455	+	AGUUGAGCUUAAAAUAAGC	19	2875
  CCR5-3456	+	AAGUUGAGCUUAAAAUAAGC	20	2876
  CCR5-3457	+	UAAGUUGAGCUUAAAAUAAGC	21	2877
  CCR5-3458	+	UUAAGUUGAGCUUAAAAUAAGC	22	2878
  CCR5-3459	+	UUUAAGUUGAGCUUAAAAUAAGC	23	2879
  CCR5-3460	+	UUUUAAGUUGAGCUUAAAAUAAGC	24	2880
  CCR5-3461	+	AAUAAAGGAUAUCAGAGC	18	2881
  CCR5-3462	+	GAAUAAAGGAUAUCAGAGC	19	2882
  CCR5-3463	+	AGAAUAAAGGAUAUCAGAGC	20	2883
  CCR5-3464	+	AAGAAUAAAGGAUAUCAGAGC	21	2884
  CCR5-3465	+	AAAGAAUAAAGGAUAUCAGAGC	22	2885
  CCR5-3466	+	UAAAGAAUAAAGGAUAUCAGAGC	23	2886
  CCR5-3467	+	AUAAAGAAUAAAGGAUAUCAGAGC	24	2887
  CCR5-3468	+	UAAAUGUAGAGGGGGAUC	18	2888
  CCR5-3469	+	UUAAAUGUAGAGGGGGAUC	19	2889
  CCR5-3470	+	UUUAAAUGUAGAGGGGGAUC	20	2890
  CCR5-3471	+	CUUUAAAUGUAGAGGGGGAUC	21	2891
  CCR5-3472	+	ACUUUAAAUGUAGAGGGGGAUC	22	2892
  CCR5-3473	+	AACUUUAAAUGUAGAGGGGGAUC	23	2893
  CCR5-3474	+	CAACUUUAAAUGUAGAGGGGGAUC	24	2894
  CCR5-3475	+	AUAUAGACAGUAUAAAAG	18	2895
  CCR5-3476	+	CAUAUAGACAGUAUAAAAG	19	2896
  CCR5-3477	+	UCAUAUAGACAGUAUAAAAG	20	2897
  CCR5-3478	+	AUCAUAUAGACAGUAUAAAAG	21	2898
  CCR5-3479	+	AAUCAUAUAGACAGUAUAAAAG	22	2899
  CCR5-3480	+	CAAUCAUAUAGACAGUAUAAAAG	23	2900
  CCR5-3481	+	UCAAUCAUAUAGACAGUAUAAAAG	24	2901
  CCR5-3482	+	UCAUUAAGUGUAUUGAAG	18	2902
  CCR5-3483	+	AUCAUUAAGUGUAUUGAAG	19	2903
  CCR5-3484	+	AAUCAUUAAGUGUAUUGAAG	20	2904
  CCR5-3485	+	AAAUCAUUAAGUGUAUUGAAG	21	2905
  CCR5-3486	+	UAAAUCAUUAAGUGUAUUGAAG	22	2906
  CCR5-3487	+	UUAAAUCAUUAAGUGUAUUGAAG	23	2907
  CCR5-3488	+	GUUAAAUCAUUAAGUGUAUUGAAG	24	2908
  CCR5-3489	+	ACAGUUCUUCUUUUUAAG	18	2909
  CCR5-3490	+	AACAGUUCUUCUUUUUAAG	19	2910
  CCR5-3491	+	GAACAGUUCUUCUUUUUAAG	20	2911
  CCR5-3492	+	AGAACAGUUCUUCUUUUUAAG	21	2912
  CCR5-3493	+	GAGAACAGUUCUUCUUUUUAAG	22	2913
  CCR5-3494	+	AGAGAACAGUUCUUCUUUUUAAG	23	2914
  CCR5-3495	+	CAGAGAACAGUUCUUCUUUUUAAG	24	2915
  CCR5-3496	+	CUCAGCUCUUCUGGCCAG	18	2916
  CCR5-3497	+	UCUCAGCUCUUCUGGCCAG	19	2917
  CCR5-3498	+	GUCUCAGCUCUUCUGGCCAG	20	2918
  CCR5-3499	+	UGUCUCAGCUCUUCUGGCCAG	21	2919
  CCR5-3500	+	AUGUCUCAGCUCUUCUGGCCAG	22	2920
  CCR5-3501	+	GAUGUCUCAGCUCUUCUGGCCAG	23	2921
  CCR5-3502	+	GGAUGUCUCAGCUCUUCUGGCCAG	24	2922
  CCR5-3503	+	AACUAACAGGCCAAGCAG	18	2923
  CCR5-3504	+	UAACUAACAGGCCAAGCAG	19	2924
  CCR5-3505	+	CUAACUAACAGGCCAAGCAG	20	2925
  CCR5-3506	+	GCUAACUAACAGGCCAAGCAG	21	2926
  CCR5-3507	+	AGCUAACUAACAGGCCAAGCAG	22	2927
  CCR5-3508	+	AAGCUAACUAACAGGCCAAGCAG	23	2928
  CCR5-3509	+	GAAGCUAACUAACAGGCCAAGCAG	24	2929
  CCR5-3510	+	AAAGGAUAUCAGAGCUAG	18	2930
  CCR5-3511	+	UAAAGGAUAUCAGAGCUAG	19	2931
  CCR5-3512	+	AUAAAGGAUAUCAGAGCUAG	20	2932
  CCR5-3513	+	AAUAAAGGAUAUCAGAGCUAG	21	2933
  CCR5-3514	+	GAAUAAAGGAUAUCAGAGCUAG	22	2934
  CCR5-3515	+	AGAAUAAAGGAUAUCAGAGCUAG	23	2935
  CCR5-3516	+	AAGAAUAAAGGAUAUCAGAGCUAG	24	2936
  CCR5-3517	+	AACCAACUUUAAAUGUAG	18	2937
  CCR5-3518	+	AAACCAACUUUAAAUGUAG	19	2938
  CCR5-2949	+	UAAACCAACUUUAAAUGUAG	20	2939
  CCR5-3519	+	UUAAACCAACUUUAAAUGUAG	21	2940
  CCR5-3520	+	CUUAAACCAACUUUAAAUGUAG	22	2941
  CCR5-3521	+	ACUUAAACCAACUUUAAAUGUAG	23	2942
  CCR5-3522	+	AACUUAAACCAACUUUAAAUGUAG	24	2943
  CCR5-3523	+	GGGGAGAGUUUCUUGUAG	18	2944
  CCR5-3524	+	CGGGGAGAGUUUCUUGUAG	19	2945
  CCR5-2820	+	CCGGGGAGAGUUUCUUGUAG	20	2946
  CCR5-3525	+	ACCGGGGAGAGUUUCUUGUAG	21	2947
  CCR5-3526	+	UACCGGGGAGAGUUUCUUGUAG	22	2948
  CCR5-3527	+	UUACCGGGGAGAGUUUCUUGUAG	23	2949
  CCR5-3528	+	CUUACCGGGGAGAGUUUCUUGUAG	24	2950
  CCR5-3529	+	GGGUUUAGUUCUCCUUAG	18	2951
  CCR5-3530	+	AGGGUUUAGUUCUCCUUAG	19	2952
  CCR5-3531	+	GAGGGUUUAGUUCUCCUUAG	20	2953
  CCR5-3532	+	AGAGGGUUUAGUUCUCCUUAG	21	2954
  CCR5-3533	+	GAGAGGGUUUAGUUCUCCUUAG	22	2955
  CCR5-3534	+	GGAGAGGGUUUAGUUCUCCUUAG	23	2956
  CCR5-3535	+	UGGAGAGGGUUUAGUUCUCCUUAG	24	2957
  CCR5-3536	+	CUGAGAGGUUACUUACCG	18	2958
  CCR5-3537	+	GCUGAGAGGUUACUUACCG	19	2959
  CCR5-2821	+	AGCUGAGAGGUUACUUACCG	20	2960
  CCR5-3538	+	CAGCUGAGAGGUUACUUACCG	21	2961
  CCR5-3539	+	GCAGCUGAGAGGUUACUUACCG	22	2962
  CCR5-3540	+	AGCAGCUGAGAGGUUACUUACCG	23	2963
  CCR5-3541	+	AAGCAGCUGAGAGGUUACUUACCG	24	2964
  CCR5-3542	+	UGUUUCUUUUGAAGGAGG	18	2965
  CCR5-3543	+	CUGUUUCUUUUGAAGGAGG	19	2966
  CCR5-3544	+	GCUGUUUCUUUUGAAGGAGG	20	2967
  CCR5-3545	+	UGCUGUUUCUUUUGAAGGAGG	21	2968
  CCR5-3546	+	AUGCUGUUUCUUUUGAAGGAGG	22	2969
  CCR5-3547	+	AAUGCUGUUUCUUUUGAAGGAGG	23	2970
  CCR5-3548	+	AAAUGCUGUUUCUUUUGAAGGAGG	24	2971
  CCR5-3549	+	UUAAACCAACUUUAAAUG	18	2972
  CCR5-3550	+	CUUAAACCAACUUUAAAUG	19	2973
  CCR5-3551	+	ACUUAAACCAACUUUAAAUG	20	2974
  CCR5-3552	+	AACUUAAACCAACUUUAAAUG	21	2975
  CCR5-3553	+	CAACUUAAACCAACUUUAAAUG	22	2976
  CCR5-3554	+	CCAACUUAAACCAACUUUAAAUG	23	2977
  CCR5-3555	+	GCCAACUUAAACCAACUUUAAAUG	24	2978
  CCR5-3556	+	UCAGAAGGCAUCUCACUG	18	2979
  CCR5-3557	+	UUCAGAAGGCAUCUCACUG	19	2980
  CCR5-3558	+	AUUCAGAAGGCAUCUCACUG	20	2981
  CCR5-3559	+	UAUUCAGAAGGCAUCUCACUG	21	2982
  CCR5-3560	+	AUAUUCAGAAGGCAUCUCACUG	22	2983
  CCR5-3561	+	CAUAUUCAGAAGGCAUCUCACUG	23	2984
  CCR5-3562	+	ACAUAUUCAGAAGGCAUCUCACUG	24	2985
  CCR5-3563	+	ACCGGGGAGAGUUUCUUG	18	2986
  CCR5-3564	+	UACCGGGGAGAGUUUCUUG	19	2987
  CCR5-3565	+	UUACCGGGGAGAGUUUCUUG	20	2988
  CCR5-3566	+	CUUACCGGGGAGAGUUUCUUG	21	2989
  CCR5-3567	+	ACUUACCGGGGAGAGUUUCUUG	22	2990
  CCR5-3568	+	UACUUACCGGGGAGAGUUUCUUG	23	2991
  CCR5-3569	+	UUACUUACCGGGGAGAGUUUCUUG	24	2992
  CCR5-3570	+	GAAAUGCUGUUUCUUUUG	18	2993
  CCR5-3571	+	GGAAAUGCUGUUUCUUUUG	19	2994
  CCR5-3572	+	AGGAAAUGCUGUUUCUUUUG	20	2995
  CCR5-3573	+	UAGGAAAUGCUGUUUCUUUUG	21	2996
  CCR5-3574	+	GUAGGAAAUGCUGUUUCUUUUG	22	2997
  CCR5-3575	+	AGUAGGAAAUGCUGUUUCUUUUG	23	2998
  CCR5-3576	+	AAGUAGGAAAUGCUGUUUCUUUUG	24	2999
  CCR5-3577	+	AUUGAAGGCGAAAAGAAU	18	3000
  CCR5-3578	+	UAUUGAAGGCGAAAAGAAU	19	3001
  CCR5-3579	+	GUAUUGAAGGCGAAAAGAAU	20	3002
  CCR5-3580	+	UGUAUUGAAGGCGAAAAGAAU	21	3003
  CCR5-3581	+	GUGUAUUGAAGGCGAAAAGAAU	22	3004
  CCR5-3582	+	AGUGUAUUGAAGGCGAAAAGAAU	23	3005
  CCR5-3583	+	AAGUGUAUUGAAGGCGAAAAGAAU	24	3006
  CCR5-3584	+	AUAAAGAAUAAAGGAUAU	18	3007
  CCR5-3585	+	UAUAAAGAAUAAAGGAUAU	19	3008
  CCR5-3586	+	AUAUAAAGAAUAAAGGAUAU	20	3009
  CCR5-3587	+	AAUAUAAAGAAUAAAGGAUAU	21	3010
  CCR5-3588	+	AAAUAUAAAGAAUAAAGGAUAU	22	3011
  CCR5-3589	+	AAAAUAUAAAGAAUAAAGGAUAU	23	3012
  CCR5-3590	+	GAAAAUAUAAAGAAUAAAGGAUAU	24	3013
  CCR5-3591	+	CUAACAGGCCAAGCAGCU	18	3014
  CCR5-3592	+	ACUAACAGGCCAAGCAGCU	19	3015
  CCR5-3593	+	AACUAACAGGCCAAGCAGCU	20	3016
  CCR5-3594	+	UAACUAACAGGCCAAGCAGCU	21	3017
  CCR5-3595	+	CUAACUAACAGGCCAAGCAGCU	22	3018
  CCR5-3596	+	GCUAACUAACAGGCCAAGCAGCU	23	3019
  CCR5-3597	+	AGCUAACUAACAGGCCAAGCAGCU	24	3020
  CCR5-3598	+	AAAGUCUUUUACUCAUCU	18	3021
  CCR5-3599	+	UAAAGUCUUUUACUCAUCU	19	3022
  CCR5-3600	+	GUAAAGUCUUUUACUCAUCU	20	3023
  CCR5-3601	+	UGUAAAGUCUUUUACUCAUCU	21	3024
  CCR5-3602	+	CUGUAAAGUCUUUUACUCAUCU	22	3025
  CCR5-3603	+	CCUGUAAAGUCUUUUACUCAUCU	23	3026
  CCR5-3604	+	UCCUGUAAAGUCUUUUACUCAUCU	24	3027
  CCR5-3605	+	UAUAGACAGUAUAAAAGU	18	3028
  CCR5-3606	+	AUAUAGACAGUAUAAAAGU	19	3029
  CCR5-2967	+	CAUAUAGACAGUAUAAAAGU	20	3030
  CCR5-3607	+	UCAUAUAGACAGUAUAAAAGU	21	3031
  CCR5-3608	+	AUCAUAUAGACAGUAUAAAAGU	22	3032
  CCR5-3609	+	AAUCAUAUAGACAGUAUAAAAGU	23	3033
  CCR5-3610	+	CAAUCAUAUAGACAGUAUAAAAGU	24	3034
  CCR5-3611	+	CUUUGAUGUUAUAACCGU	18	3035
  CCR5-3612	+	UCUUUGAUGUUAUAACCGU	19	3036
  CCR5-3613	+	AUCUUUGAUGUUAUAACCGU	20	3037
  CCR5-3614	+	UAUCUUUGAUGUUAUAACCGU	21	3038
  CCR5-3615	+	GUAUCUUUGAUGUUAUAACCGU	22	3039
  CCR5-3616	+	UGUAUCUUUGAUGUUAUAACCGU	23	3040
  CCR5-3617	+	UUGUAUCUUUGAUGUUAUAACCGU	24	3041
  CCR5-3618	+	AGAGAAUAGAUCUCUGGU	18	3042
  CCR5-3619	+	UAGAGAAUAGAUCUCUGGU	19	3043
  CCR5-3620	+	CUAGAGAAUAGAUCUCUGGU	20	3044
  CCR5-3621	+	GCUAGAGAAUAGAUCUCUGGU	21	3045
  CCR5-3622	+	AGCUAGAGAAUAGAUCUCUGGU	22	3046
  CCR5-3623	+	AAGCUAGAGAAUAGAUCUCUGGU	23	3047
  CCR5-3624	+	UAAGCUAGAGAAUAGAUCUCUGGU	24	3048
  CCR5-3625	+	CCACUACACAGAAUCUGU	18	3049
  CCR5-3626	+	CCCACUACACAGAAUCUGU	19	3050
  CCR5-3627	+	UCCCACUACACAGAAUCUGU	20	3051
  CCR5-3628	+	AUCCCACUACACAGAAUCUGU	21	3052
  CCR5-3629	+	CAUCCCACUACACAGAAUCUGU	22	3053
  CCR5-3630	+	UCAUCCCACUACACAGAAUCUGU	23	3054
  CCR5-3631	+	CUCAUCCCACUACACAGAAUCUGU	24	3055
  CCR5-3632	+	AUAUUUUAAGAUAAUUGU	18	3056
  CCR5-3633	+	UAUAUUUUAAGAUAAUUGU	19	3057
  CCR5-3634	+	UUAUAUUUUAAGAUAAUUGU	20	3058
  CCR5-3635	+	AUUAUAUUUUAAGAUAAUUGU	21	3059
  CCR5-3636	+	GAUUAUAUUUUAAGAUAAUUGU	22	3060
  CCR5-3637	+	AGAUUAUAUUUUAAGAUAAUUGU	23	3061
  CCR5-3638	+	AAGAUUAUAUUUUAAGAUAAUUGU	24	3062
  CCR5-3639	+	CCGGGGAGAGUUUCUUGU	18	3063
  CCR5-3640	+	ACCGGGGAGAGUUUCUUGU	19	3064
  CCR5-2974	+	UACCGGGGAGAGUUUCUUGU	20	3065
  CCR5-3641	+	UUACCGGGGAGAGUUUCUUGU	21	3066
  CCR5-3642	+	CUUACCGGGGAGAGUUUCUUGU	22	3067
  CCR5-3643	+	ACUUACCGGGGAGAGUUUCUUGU	23	3068
  CCR5-3644	+	UACUUACCGGGGAGAGUUUCUUGU	24	3069
  CCR5-3645	+	UCUCUGCAAAUCUUUCUU	18	3070
  CCR5-3646	+	CUCUCUGCAAAUCUUUCUU	19	3071
  CCR5-3647	+	UCUCUCUGCAAAUCUUUCUU	20	3072
  CCR5-3648	+	AUCUCUCUGCAAAUCUUUCUU	21	3073
  CCR5-3649	+	CAUCUCUCUGCAAAUCUUUCUU	22	3074
  CCR5-3650	+	UCAUCUCUCUGCAAAUCUUUCUU	23	3075
  CCR5-3651	+	CUCAUCUCUCUGCAAAUCUUUCUU	24	3076
  CCR5-3652	+	UAGGAAAUGCUGUUUCUU	18	3077
  CCR5-3653	+	GUAGGAAAUGCUGUUUCUU	19	3078
  CCR5-3654	+	AGUAGGAAAUGCUGUUUCUU	20	3079
  CCR5-3655	+	AAGUAGGAAAUGCUGUUUCUU	21	3080
  CCR5-3656	+	AAAGUAGGAAAUGCUGUUUCUU	22	3081
  CCR5-3657	+	AAAAGUAGGAAAUGCUGUUUCUU	23	3082
  CCR5-3658	+	UAAAAGUAGGAAAUGCUGUUUCUU	24	3083
  CCR5-3659	+	CAGUAAGGCUAAAAGGUU	18	3084
  CCR5-3660	+	ACAGUAAGGCUAAAAGGUU	19	3085
  CCR5-3661	+	AACAGUAAGGCUAAAAGGUU	20	3086
  CCR5-3662	+	CAACAGUAAGGCUAAAAGGUU	21	3087
  CCR5-3663	+	UCAACAGUAAGGCUAAAAGGUU	22	3088
  CCR5-3664	+	UUCAACAGUAAGGCUAAAAGGUU	23	3089
  CCR5-3665	+	UUUCAACAGUAAGGCUAAAAGGUU	24	3090
  CCR5-3666	+	UGGUCUGAAGGUUUAUUU	18	3091
  CCR5-3667	+	CUGGUCUGAAGGUUUAUUU	19	3092
  CCR5-3668	+	UCUGGUCUGAAGGUUUAUUU	20	3093
  CCR5-3669	+	CUCUGGUCUGAAGGUUUAUUU	21	3094
  CCR5-3670	+	UCUCUGGUCUGAAGGUUUAUUU	22	3095
  CCR5-3671	+	AUCUCUGGUCUGAAGGUUUAUUU	23	3096
  CCR5-3672	+	GAUCUCUGGUCUGAAGGUUUAUUU	24	3097
  CCR5-3673	+	UCUGCAAAUCUUUCUUUU	18	3098
  CCR5-3674	+	CUCUGCAAAUCUUUCUUUU	19	3099
  CCR5-3675	+	UCUCUGCAAAUCUUUCUUUU	20	3100
  CCR5-3676	+	CUCUCUGCAAAUCUUUCUUUU	21	3101
  CCR5-3677	+	UCUCUCUGCAAAUCUUUCUUUU	22	3102
  CCR5-3678	+	AUCUCUCUGCAAAUCUUUCUUUU	23	3103
  CCR5-3679	+	CAUCUCUCUGCAAAUCUUUCUUUU	24	3104
  CCR5-3680	−	GGGGAGAGUGGAGAAAAA	18	3105
  CCR5-3681	−	CGGGGAGAGUGGAGAAAAA	19	3106
  CCR5-2905	−	ACGGGGAGAGUGGAGAAAAA	20	3107
  CCR5-3682	−	UACGGGGAGAGUGGAGAAAAA	21	3108
  CCR5-3683	−	AUACGGGGAGAGUGGAGAAAAA	22	3109
  CCR5-3684	−	GAUACGGGGAGAGUGGAGAAAAA	23	3110
  CCR5-3685	−	GGAUACGGGGAGAGUGGAGAAAAA	24	3111
  CCR5-3686	−	CGGGGAGAGUGGAGAAAA	18	3112
  CCR5-3687	−	ACGGGGAGAGUGGAGAAAA	19	3113
  CCR5-2906	−	UACGGGGAGAGUGGAGAAAA	20	3114
  CCR5-3688	−	AUACGGGGAGAGUGGAGAAAA	21	3115
  CCR5-3689	−	GAUACGGGGAGAGUGGAGAAAA	22	3116
  CCR5-3690	−	GGAUACGGGGAGAGUGGAGAAAA	23	3117
  CCR5-3691	−	GGGAUACGGGGAGAGUGGAGAAAA	24	3118
  CCR5-3692	−	ACGGGGAGAGUGGAGAAA	18	3119
  CCR5-3693	−	UACGGGGAGAGUGGAGAAA	19	3120
  CCR5-3694	−	AUACGGGGAGAGUGGAGAAA	20	3121
  CCR5-3695	−	GAUACGGGGAGAGUGGAGAAA	21	3122
  CCR5-3696	−	GGAUACGGGGAGAGUGGAGAAA	22	3123
  CCR5-3697	−	GGGAUACGGGGAGAGUGGAGAAA	23	3124
  CCR5-3698	−	GGGGAUACGGGGAGAGUGGAGAAA	24	3125
  CCR5-3699	−	UUUUAAGCUCAACUUAAA	18	3126
  CCR5-3700	−	AUUUUAAGCUCAACUUAAA	19	3127
  CCR5-3701	−	UAUUUUAAGCUCAACUUAAA	20	3128
  CCR5-3702	−	UUAUUUUAAGCUCAACUUAAA	21	3129
  CCR5-3703	−	CUUAUUUUAAGCUCAACUUAAA	22	3130
  CCR5-3704	−	GCUUAUUUUAAGCUCAACUUAAA	23	3131
  CCR5-3705	−	AGCUUAUUUUAAGCUCAACUUAAA	24	3132
  CCR5-3706	−	UGAGUGAAAGACUUUAAA	18	3133
  CCR5-3707	−	GUGAGUGAAAGACUUUAAA	19	3134
  CCR5-2909	−	UGUGAGUGAAAGACUUUAAA	20	3135
  CCR5-3708	−	UUGUGAGUGAAAGACUUUAAA	21	3136
  CCR5-3709	−	AUUGUGAGUGAAAGACUUUAAA	22	3137
  CCR5-3710	−	GAUUGUGAGUGAAAGACUUUAAA	23	3138
  CCR5-3711	−	UGAUUGUGAGUGAAAGACUUUAAA	24	3139
  CCR5-3712	−	ACAAUCCUUACCUCUCAA	18	3140
  CCR5-3713	−	AACAAUCCUUACCUCUCAA	19	3141
  CCR5-3714	−	UAACAAUCCUUACCUCUCAA	20	3142
  CCR5-3715	−	CUAACAAUCCUUACCUCUCAA	21	3143
  CCR5-3716	−	ACUAACAAUCCUUACCUCUCAA	22	3144
  CCR5-3717	−	AACUAACAAUCCUUACCUCUCAA	23	3145
  CCR5-3718	−	UAACUAACAAUCCUUACCUCUCAA	24	3146
  CCR5-3719	−	AACUCCACCCUCCUUCAA	18	3147
  CCR5-3720	−	UAACUCCACCCUCCUUCAA	19	3148
  CCR5-3721	−	UUAACUCCACCCUCCUUCAA	20	3149
  CCR5-3722	−	UUUAACUCCACCCUCCUUCAA	21	3150
  CCR5-3723	−	AUUUAACUCCACCCUCCUUCAA	22	3151
  CCR5-3724	−	GAUUUAACUCCACCCUCCUUCAA	23	3152
  CCR5-3725	−	UGAUUUAACUCCACCCUCCUUCAA	24	3153
  CCR5-3726	−	GUGAGUGAAAGACUUUAA	18	3154
  CCR5-3727	−	UGUGAGUGAAAGACUUUAA	19	3155
  CCR5-2913	−	UUGUGAGUGAAAGACUUUAA	20	3156
  CCR5-3728	−	AUUGUGAGUGAAAGACUUUAA	21	3157
  CCR5-3729	−	GAUUGUGAGUGAAAGACUUUAA	22	3158
  CCR5-3730	−	UGAUUGUGAGUGAAAGACUUUAA	23	3159
  CCR5-3731	−	AUGAUUGUGAGUGAAAGACUUUAA	24	3160
  CCR5-3732	−	GACUUUACAGGAAACCCA	18	3161
  CCR5-3733	−	AGACUUUACAGGAAACCCA	19	3162
  CCR5-3734	−	AAGACUUUACAGGAAACCCA	20	3163
  CCR5-3735	−	AAAGACUUUACAGGAAACCCA	21	3164
  CCR5-3736	−	AAAAGACUUUACAGGAAACCCA	22	3165
  CCR5-3737	−	UAAAAGACUUUACAGGAAACCCA	23	3166
  CCR5-3738	−	GUAAAAGACUUUACAGGAAACCCA	24	3167
  CCR5-3739	−	CAAAAACAAAAUAAUCCA	18	3168
  CCR5-3740	−	ACAAAAACAAAAUAAUCCA	19	3169
  CCR5-3741	−	AACAAAAACAAAAUAAUCCA	20	3170
  CCR5-3742	−	GAACAAAAACAAAAUAAUCCA	21	3171
  CCR5-3743	−	AGAACAAAAACAAAAUAAUCCA	22	3172
  CCR5-3744	−	GAGAACAAAAACAAAAUAAUCCA	23	3173
  CCR5-3745	−	AGAGAACAAAAACAAAAUAAUCCA	24	3174
  CCR5-3746	−	AGAACUAAACCCUCUCCA	18	3175
  CCR5-3747	−	GAGAACUAAACCCUCUCCA	19	3176
  CCR5-3748	−	GGAGAACUAAACCCUCUCCA	20	3177
  CCR5-3749	−	AGGAGAACUAAACCCUCUCCA	21	3178
  CCR5-3750	−	AAGGAGAACUAAACCCUCUCCA	22	3179
  CCR5-3751	−	UAAGGAGAACUAAACCCUCUCCA	23	3180
  CCR5-3752	−	CUAAGGAGAACUAAACCCUCUCCA	24	3181
  CCR5-3753	−	UGUGUAGUGGGAUGAGCA	18	3182
  CCR5-3754	−	CUGUGUAGUGGGAUGAGCA	19	3183
  CCR5-3755	−	UCUGUGUAGUGGGAUGAGCA	20	3184
  CCR5-3756	−	UUCUGUGUAGUGGGAUGAGCA	21	3185
  CCR5-3757	−	AUUCUGUGUAGUGGGAUGAGCA	22	3186
  CCR5-3758	−	GAUUCUGUGUAGUGGGAUGAGCA	23	3187
  CCR5-3759	−	AGAUUCUGUGUAGUGGGAUGAGCA	24	3188
  CCR5-3760	−	UCAAAAGAAAGAUUUGCA	18	3189
  CCR5-3761	−	CUCAAAAGAAAGAUUUGCA	19	3190
  CCR5-3762	−	UCUCAAAAGAAAGAUUUGCA	20	3191
  CCR5-3763	−	CUCUCAAAAGAAAGAUUUGCA	21	3192
  CCR5-3764	−	CCUCUCAAAAGAAAGAUUUGCA	22	3193
  CCR5-3765	−	ACCUCUCAAAAGAAAGAUUUGCA	23	3194
  CCR5-3766	−	UACCUCUCAAAAGAAAGAUUUGCA	24	3195
  CCR5-3767	−	AUAGGGGAUACGGGGAGA	18	3196
  CCR5-3768	−	GAUAGGGGAUACGGGGAGA	19	3197
  CCR5-3769	−	GGAUAGGGGAUACGGGGAGA	20	3198
  CCR5-3770	−	GGGAUAGGGGAUACGGGGAGA	21	3199
  CCR5-3771	−	UGGGAUAGGGGAUACGGGGAGA	22	3200
  CCR5-3772	−	GUGGGAUAGGGGAUACGGGGAGA	23	3201
  CCR5-3773	−	GGUGGGAUAGGGGAUACGGGGAGA	24	3202
  CCR5-3774	−	GUGGGGGUUGGGGUGGGA	18	3203
  CCR5-3775	−	UGUGGGGGUUGGGGUGGGA	19	3204
  CCR5-3776	−	GUGUGGGGGUUGGGGUGGGA	20	3205
  CCR5-3777	−	UGUGUGGGGGUUGGGGUGGGA	21	3206
  CCR5-3778	−	CUGUGUGGGGGUUGGGGUGGGA	22	3207
  CCR5-3779	−	UCUGUGUGGGGGUUGGGGUGGGA	23	3208
  CCR5-3780	−	AUCUGUGUGGGGGUUGGGGUGGGA	24	3209
  CCR5-3781	−	UACAAAACAUGAUUGUGA	18	3210
  CCR5-3782	−	AUACAAAACAUGAUUGUGA	19	3211
  CCR5-3783	−	GAUACAAAACAUGAUUGUGA	20	3212
  CCR5-3784	−	AGAUACAAAACAUGAUUGUGA	21	3213
  CCR5-3785	−	AAGAUACAAAACAUGAUUGUGA	22	3214
  CCR5-3786	−	AAAGAUACAAAACAUGAUUGUGA	23	3215
  CCR5-3787	−	CAAAGAUACAAAACAUGAUUGUGA	24	3216
  CCR5-3788	−	AAUAUAAUCUUUAAGAUA	18	3217
  CCR5-3789	−	AAAUAUAAUCUUUAAGAUA	19	3218
  CCR5-2922	−	AAAAUAUAAUCUUUAAGAUA	20	3219
  CCR5-3790	−	UAAAAUAUAAUCUUUAAGAUA	21	3220
  CCR5-3791	−	UUAAAAUAUAAUCUUUAAGAUA	22	3221
  CCR5-3792	−	CUUAAAAUAUAAUCUUUAAGAUA	23	3222
  CCR5-3793	−	UCUUAAAAUAUAAUCUUUAAGAUA	24	3223
  CCR5-3794	−	GGGGUGGGAUAGGGGAUA	18	3224
  CCR5-3795	−	UGGGGUGGGAUAGGGGAUA	19	3225
  CCR5-2923	−	UUGGGGUGGGAUAGGGGAUA	20	3226
  CCR5-3796	−	GUUGGGGUGGGAUAGGGGAUA	21	3227
  CCR5-3797	−	GGUUGGGGUGGGAUAGGGGAUA	22	3228
  CCR5-3798	−	GGGUUGGGGUGGGAUAGGGGAUA	23	3229
  CCR5-3799	−	GGGGUUGGGGUGGGAUAGGGGAUA	24	3230
  CCR5-3800	−	AAAUCUUAUCUUCUGCUA	18	3231
  CCR5-3801	−	GAAAUCUUAUCUUCUGCUA	19	3232
  CCR5-2925	−	UGAAAUCUUAUCUUCUGCUA	20	3233
  CCR5-3802	−	UUGAAAUCUUAUCUUCUGCUA	21	3234
  CCR5-3803	−	CUUGAAAUCUUAUCUUCUGCUA	22	3235
  CCR5-3804	−	UCUUGAAAUCUUAUCUUCUGCUA	23	3236
  CCR5-3805	−	AUCUUGAAAUCUUAUCUUCUGCUA	24	3237
  CCR5-3806	−	UCUAACAGAUUCUGUGUA	18	3238
  CCR5-3807	−	UUCUAACAGAUUCUGUGUA	19	3239
  CCR5-3808	−	UUUCUAACAGAUUCUGUGUA	20	3240
  CCR5-3809	−	UUUUCUAACAGAUUCUGUGUA	21	3241
  CCR5-3810	−	AUUUUCUAACAGAUUCUGUGUA	22	3242
  CCR5-3811	−	UAUUUUCUAACAGAUUCUGUGUA	23	3243
  CCR5-3812	−	AUAUUUUCUAACAGAUUCUGUGUA	24	3244
  CCR5-3813	−	GAUGAGUAAAAGACUUUA	18	3245
  CCR5-3814	−	AGAUGAGUAAAAGACUUUA	19	3246
  CCR5-3815	−	GAGAUGAGUAAAAGACUUUA	20	3247
  CCR5-3816	−	UGAGAUGAGUAAAAGACUUUA	21	3248
  CCR5-3817	−	CUGAGAUGAGUAAAAGACUUUA	22	3249
  CCR5-3818	−	UCUGAGAUGAGUAAAAGACUUUA	23	3250
  CCR5-3819	−	UUCUGAGAUGAGUAAAAGACUUUA	24	3251
  CCR5-3820	−	UGUGAGUGAAAGACUUUA	18	3252
  CCR5-3821	−	UUGUGAGUGAAAGACUUUA	19	3253
  CCR5-3822	−	AUUGUGAGUGAAAGACUUUA	20	3254
  CCR5-3823	−	GAUUGUGAGUGAAAGACUUUA	21	3255
  CCR5-3824	−	UGAUUGUGAGUGAAAGACUUUA	22	3256
  CCR5-3825	−	AUGAUUGUGAGUGAAAGACUUUA	23	3257
  CCR5-3826	−	CAUGAUUGUGAGUGAAAGACUUUA	24	3258
  CCR5-3827	−	GUAAAUAAACCUUCAGAC	18	3259
  CCR5-3828	−	CGUAAAUAAACCUUCAGAC	19	3260
  CCR5-3829	−	CCGUAAAUAAACCUUCAGAC	20	3261
  CCR5-3830	−	CCCGUAAAUAAACCUUCAGAC	21	3262
  CCR5-3831	−	GCCCGUAAAUAAACCUUCAGAC	22	3263
  CCR5-3832	−	AGCCCGUAAAUAAACCUUCAGAC	23	3264
  CCR5-3833	−	AAGCCCGUAAAUAAACCUUCAGAC	24	3265
  CCR5-3834	−	GGGUGGGAUAGGGGAUAC	18	3266
  CCR5-3835	−	GGGGUGGGAUAGGGGAUAC	19	3267
  CCR5-2934	−	UGGGGUGGGAUAGGGGAUAC	20	3268
  CCR5-3836	−	UUGGGGUGGGAUAGGGGAUAC	21	3269
  CCR5-3837	−	GUUGGGGUGGGAUAGGGGAUAC	22	3270
  CCR5-3838	−	GGUUGGGGUGGGAUAGGGGAUAC	23	3271
  CCR5-3839	−	GGGUUGGGGUGGGAUAGGGGAUAC	24	3272
  CCR5-3840	−	AGACAUCCGUUCCCCUAC	18	3273
  CCR5-3841	−	GAGACAUCCGUUCCCCUAC	19	3274
  CCR5-3842	−	UGAGACAUCCGUUCCCCUAC	20	3275
  CCR5-3843	−	CUGAGACAUCCGUUCCCCUAC	21	3276
  CCR5-3844	−	GCUGAGACAUCCGUUCCCCUAC	22	3277
  CCR5-3845	−	AGCUGAGACAUCCGUUCCCCUAC	23	3278
  CCR5-3846	−	GAGCUGAGACAUCCGUUCCCCUAC	24	3279
  CCR5-3847	−	AUGAGUAAAAGACUUUAC	18	3280
  CCR5-3848	−	GAUGAGUAAAAGACUUUAC	19	3281
  CCR5-2936	−	AGAUGAGUAAAAGACUUUAC	20	3282
  CCR5-3849	−	GAGAUGAGUAAAAGACUUUAC	21	3283
  CCR5-3850	−	UGAGAUGAGUAAAAGACUUUAC	22	3284
  CCR5-3851	−	CUGAGAUGAGUAAAAGACUUUAC	23	3285
  CCR5-3852	−	UCUGAGAUGAGUAAAAGACUUUAC	24	3286
  CCR5-3853	−	UUGCACAGCUCAUCUGGC	18	3287
  CCR5-3854	−	UUUGCACAGCUCAUCUGGC	19	3288
  CCR5-3855	−	AUUUGCACAGCUCAUCUGGC	20	3289
  CCR5-3856	−	GAUUUGCACAGCUCAUCUGGC	21	3290
  CCR5-3857	−	UGAUUUGCACAGCUCAUCUGGC	22	3291
  CCR5-3858	−	UUGAUUUGCACAGCUCAUCUGGC	23	3292
  CCR5-3859	−	AUUGAUUUGCACAGCUCAUCUGGC	24	3293
  CCR5-3860	−	UGAGUCUUAGCUGAAAUC	18	3294
  CCR5-3861	−	AUGAGUCUUAGCUGAAAUC	19	3295
  CCR5-3862	−	GAUGAGUCUUAGCUGAAAUC	20	3296
  CCR5-3863	−	AGAUGAGUCUUAGCUGAAAUC	21	3297
  CCR5-3864	−	GAGAUGAGUCUUAGCUGAAAUC	22	3298
  CCR5-3865	−	AGAGAUGAGUCUUAGCUGAAAUC	23	3299
  CCR5-3866	−	GAGAGAUGAGUCUUAGCUGAAAUC	24	3300
  CCR5-3867	−	UAAGCUCAACUUAAAAAG	18	3301
  CCR5-3868	−	UUAAGCUCAACUUAAAAAG	19	3302
  CCR5-3869	−	UUUAAGCUCAACUUAAAAAG	20	3303
  CCR5-3870	−	UUUUAAGCUCAACUUAAAAAG	21	3304
  CCR5-3871	−	AUUUUAAGCUCAACUUAAAAAG	22	3305
  CCR5-3872	−	UAUUUUAAGCUCAACUUAAAAAG	23	3306
  CCR5-3873	−	UUAUUUUAAGCUCAACUUAAAAAG	24	3307
  CCR5-3874	−	AUCUUAUCUUCUGCUAAG	18	3308
  CCR5-3875	−	AAUCUUAUCUUCUGCUAAG	19	3309
  CCR5-3876	−	AAAUCUUAUCUUCUGCUAAG	20	3310
  CCR5-3877	−	GAAAUCUUAUCUUCUGCUAAG	21	3311
  CCR5-3878	−	UGAAAUCUUAUCUUCUGCUAAG	22	3312
  CCR5-3879	−	UUGAAAUCUUAUCUUCUGCUAAG	23	3313
  CCR5-3880	−	CUUGAAAUCUUAUCUUCUGCUAAG	24	3314
  CCR5-3881	−	CACAGCUCAUCUGGCCAG	18	3315
  CCR5-3882	−	GCACAGCUCAUCUGGCCAG	19	3316
  CCR5-3883	−	UGCACAGCUCAUCUGGCCAG	20	3317
  CCR5-3884	−	UUGCACAGCUCAUCUGGCCAG	21	3318
  CCR5-3885	−	UUUGCACAGCUCAUCUGGCCAG	22	3319
  CCR5-3886	−	AUUUGCACAGCUCAUCUGGCCAG	23	3320
  CCR5-3887	−	GAUUUGCACAGCUCAUCUGGCCAG	24	3321
  CCR5-3888	−	CUCAUCUGGCCAGAAGAG	18	3322
  CCR5-3889	−	GCUCAUCUGGCCAGAAGAG	19	3323
  CCR5-3890	−	AGCUCAUCUGGCCAGAAGAG	20	3324
  CCR5-3891	−	CAGCUCAUCUGGCCAGAAGAG	21	3325
  CCR5-3892	−	ACAGCUCAUCUGGCCAGAAGAG	22	3326
  CCR5-3893	−	CACAGCUCAUCUGGCCAGAAGAG	23	3327
  CCR5-3894	−	GCACAGCUCAUCUGGCCAGAAGAG	24	3328
  CCR5-3895	−	UAGGGGAUACGGGGAGAG	18	3329
  CCR5-3896	−	AUAGGGGAUACGGGGAGAG	19	3330
  CCR5-2819	−	GAUAGGGGAUACGGGGAGAG	20	3331
  CCR5-3897	−	GGAUAGGGGAUACGGGGAGAG	21	3332
  CCR5-3898	−	GGGAUAGGGGAUACGGGGAGAG	22	3333
  CCR5-3899	−	UGGGAUAGGGGAUACGGGGAGAG	23	3334
  CCR5-3900	−	GUGGGAUAGGGGAUACGGGGAGAG	24	3335
  CCR5-3901	−	UCUGUGUAGUGGGAUGAG	18	3336
  CCR5-3902	−	UUCUGUGUAGUGGGAUGAG	19	3337
  CCR5-3903	−	AUUCUGUGUAGUGGGAUGAG	20	3338
  CCR5-3904	−	GAUUCUGUGUAGUGGGAUGAG	21	3339
  CCR5-3905	−	AGAUUCUGUGUAGUGGGAUGAG	22	3340
  CCR5-3906	−	CAGAUUCUGUGUAGUGGGAUGAG	23	3341
  CCR5-3907	−	ACAGAUUCUGUGUAGUGGGAUGAG	24	3342
  CCR5-3908	−	CAGAGAGAUGAGUCUUAG	18	3343
  CCR5-3909	−	GCAGAGAGAUGAGUCUUAG	19	3344
  CCR5-3910	−	UGCAGAGAGAUGAGUCUUAG	20	3345
  CCR5-3911	−	UUGCAGAGAGAUGAGUCUUAG	21	3346
  CCR5-3912	−	UUUGCAGAGAGAUGAGUCUUAG	22	3347
  CCR5-3913	−	AUUUGCAGAGAGAUGAGUCUUAG	23	3348
  CCR5-3914	−	GAUUUGCAGAGAGAUGAGUCUUAG	24	3349
  CCR5-3915	−	GGUGGGAUAGGGGAUACG	18	3350
  CCR5-3916	−	GGGUGGGAUAGGGGAUACG	19	3351
  CCR5-2951	−	GGGGUGGGAUAGGGGAUACG	20	3352
  CCR5-3917	−	UGGGGUGGGAUAGGGGAUACG	21	3353
  CCR5-3918	−	UUGGGGUGGGAUAGGGGAUACG	22	3354
  CCR5-3919	−	GUUGGGGUGGGAUAGGGGAUACG	23	3355
  CCR5-3920	−	GGUUGGGGUGGGAUAGGGGAUACG	24	3356
  CCR5-3921	−	UGAGCAUCUGUGUGGGGG	18	3357
  CCR5-3922	−	GUGAGCAUCUGUGUGGGGG	19	3358
  CCR5-3923	−	GGUGAGCAUCUGUGUGGGGG	20	3359
  CCR5-3924	−	UGGUGAGCAUCUGUGUGGGGG	21	3360
  CCR5-3925	−	GUGGUGAGCAUCUGUGUGGGGG	22	3361
  CCR5-3926	−	GGUGGUGAGCAUCUGUGUGGGGG	23	3362
  CCR5-3927	−	GGGUGGUGAGCAUCUGUGUGGGGG	24	3363
  CCR5-3928	−	CAGAUUCUGUGUAGUGGG	18	3364
  CCR5-3929	−	ACAGAUUCUGUGUAGUGGG	19	3365
  CCR5-3930	−	AACAGAUUCUGUGUAGUGGG	20	3366
  CCR5-3931	−	UAACAGAUUCUGUGUAGUGGG	21	3367
  CCR5-3932	−	CUAACAGAUUCUGUGUAGUGGG	22	3368
  CCR5-3933	−	UCUAACAGAUUCUGUGUAGUGGG	23	3369
  CCR5-3934	−	UUCUAACAGAUUCUGUGUAGUGGG	24	3370
  CCR5-3935	−	AUCUGUGUGGGGGUUGGG	18	3371
  CCR5-3936	−	CAUCUGUGUGGGGGUUGGG	19	3372
  CCR5-3937	−	GCAUCUGUGUGGGGGUUGGG	20	3373
  CCR5-3938	−	AGCAUCUGUGUGGGGGUUGGG	21	3374
  CCR5-3939	−	GAGCAUCUGUGUGGGGGUUGGG	22	3375
  CCR5-3940	−	UGAGCAUCUGUGUGGGGGUUGGG	23	3376
  CCR5-3941	−	GUGAGCAUCUGUGUGGGGGUUGGG	24	3377
  CCR5-3942	−	AACCUUUUAGCCUUACUG	18	3378
  CCR5-3943	−	UAACCUUUUAGCCUUACUG	19	3379
  CCR5-3944	−	UUAACCUUUUAGCCUUACUG	20	3380
  CCR5-3945	−	CUUAACCUUUUAGCCUUACUG	21	3381
  CCR5-3946	−	UCUUAACCUUUUAGCCUUACUG	22	3382
  CCR5-3947	−	UUCUUAACCUUUUAGCCUUACUG	23	3383
  CCR5-3948	−	UUUCUUAACCUUUUAGCCUUACUG	24	3384
  CCR5-3949	−	GGGGAUACGGGGAGAGUG	18	3385
  CCR5-3950	−	AGGGGAUACGGGGAGAGUG	19	3386
  CCR5-3951	−	UAGGGGAUACGGGGAGAGUG	20	3387
  CCR5-3952	−	AUAGGGGAUACGGGGAGAGUG	21	3388
  CCR5-3953	−	GAUAGGGGAUACGGGGAGAGUG	22	3389
  CCR5-3954	−	GGAUAGGGGAUACGGGGAGAGUG	23	3390
  CCR5-3955	−	GGGAUAGGGGAUACGGGGAGAGUG	24	3391
  CCR5-3956	−	GAACAAUAAUAUUGGGUG	18	3392
  CCR5-3957	−	AGAACAAUAAUAUUGGGUG	19	3393
  CCR5-3958	−	GAGAACAAUAAUAUUGGGUG	20	3394
  CCR5-3959	−	AGAGAACAAUAAUAUUGGGUG	21	3395
  CCR5-3960	−	CAGAGAACAAUAAUAUUGGGUG	22	3396
  CCR5-3961	−	ACAGAGAACAAUAAUAUUGGGUG	23	3397
  CCR5-3962	−	UACAGAGAACAAUAAUAUUGGGUG	24	3398
  CCR5-3963	−	GGGUGGUGAGCAUCUGUG	18	3399
  CCR5-3964	−	UGGGUGGUGAGCAUCUGUG	19	3400
  CCR5-2959	−	UUGGGUGGUGAGCAUCUGUG	20	3401
  CCR5-3965	−	AUUGGGUGGUGAGCAUCUGUG	21	3402
  CCR5-3966	−	UAUUGGGUGGUGAGCAUCUGUG	22	3403
  CCR5-3967	−	AUAUUGGGUGGUGAGCAUCUGUG	23	3404
  CCR5-3968	−	AAUAUUGGGUGGUGAGCAUCUGUG	24	3405
  CCR5-3969	−	UCUCAAAAGAAAGAUUUG	18	3406
  CCR5-3970	−	CUCUCAAAAGAAAGAUUUG	19	3407
  CCR5-3971	−	CCUCUCAAAAGAAAGAUUUG	20	3408
  CCR5-3972	−	ACCUCUCAAAAGAAAGAUUUG	21	3409
  CCR5-3973	−	UACCUCUCAAAAGAAAGAUUUG	22	3410
  CCR5-3974	−	UUACCUCUCAAAAGAAAGAUUUG	23	3411
  CCR5-3975	−	CUUACCUCUCAAAAGAAAGAUUUG	24	3412
  CCR5-3976	−	AAUUUCUUUUACUAAAAU	18	3413
  CCR5-3977	−	UAAUUUCUUUUACUAAAAU	19	3414
  CCR5-3978	−	GUAAUUUCUUUUACUAAAAU	20	3415
  CCR5-3979	−	AGUAAUUUCUUUUACUAAAAU	21	3416
  CCR5-3980	−	UAGUAAUUUCUUUUACUAAAAU	22	3417
  CCR5-3981	−	AUAGUAAUUUCUUUUACUAAAAU	23	3418
  CCR5-3982	−	GAUAGUAAUUUCUUUUACUAAAAU	24	3419
  CCR5-3983	−	AGGGGACACAGGGUUAAU	18	3420
  CCR5-3984	−	AAGGGGACACAGGGUUAAU	19	3421
  CCR5-3985	−	AAAGGGGACACAGGGUUAAU	20	3422
  CCR5-3986	−	AAAAGGGGACACAGGGUUAAU	21	3423
  CCR5-3987	−	AAAAAGGGGACACAGGGUUAAU	22	3424
  CCR5-3988	−	GAAAAAGGGGACACAGGGUUAAU	23	3425
  CCR5-3989	−	AGAAAAAGGGGACACAGGGUUAAU	24	3426
  CCR5-3990	−	AAAUAUAAUCUUUAAGAU	18	3427
  CCR5-3991	−	AAAAUAUAAUCUUUAAGAU	19	3428
  CCR5-3992	−	UAAAAUAUAAUCUUUAAGAU	20	3429
  CCR5-3993	−	UUAAAAUAUAAUCUUUAAGAU	21	3430
  CCR5-3994	−	CUUAAAAUAUAAUCUUUAAGAU	22	3431
  CCR5-3995	−	UCUUAAAAUAUAAUCUUUAAGAU	23	3432
  CCR5-3996	−	AUCUUAAAAUAUAAUCUUUAAGAU	24	3433
  CCR5-3997	−	UGGGGUGGGAUAGGGGAU	18	3434
  CCR5-3998	−	UUGGGGUGGGAUAGGGGAU	19	3435
  CCR5-3999	−	GUUGGGGUGGGAUAGGGGAU	20	3436
  CCR5-4000	−	GGUUGGGGUGGGAUAGGGGAU	21	3437
  CCR5-4001	−	GGGUUGGGGUGGGAUAGGGGAU	22	3438
  CCR5-4002	−	GGGGUUGGGGUGGGAUAGGGGAU	23	3439
  CCR5-4003	−	GGGGGUUGGGGUGGGAUAGGGGAU	24	3440
  CCR5-4004	−	UGGGGGUUGGGGUGGGAU	18	3441
  CCR5-4005	−	GUGGGGGUUGGGGUGGGAU	19	3442
  CCR5-2962	−	UGUGGGGGUUGGGGUGGGAU	20	3443
  CCR5-4006	−	GUGUGGGGGUUGGGGUGGGAU	21	3444
  CCR5-4007	−	UGUGUGGGGGUUGGGGUGGGAU	22	3445
  CCR5-4008	−	CUGUGUGGGGGUUGGGGUGGGAU	23	3446
  CCR5-4009	−	UCUGUGUGGGGGUUGGGGUGGGAU	24	3447
  CCR5-4010	−	GAAAUCUUAUCUUCUGCU	18	3448
  CCR5-4011	−	UGAAAUCUUAUCUUCUGCU	19	3449
  CCR5-4012	−	UUGAAAUCUUAUCUUCUGCU	20	3450
  CCR5-4013	−	CUUGAAAUCUUAUCUUCUGCU	21	3451
  CCR5-4014	−	UCUUGAAAUCUUAUCUUCUGCU	22	3452
  CCR5-4015	−	AUCUUGAAAUCUUAUCUUCUGCU	23	3453
  CCR5-4016	−	AAUCUUGAAAUCUUAUCUUCUGCU	24	3454
  CCR5-4017	−	UAAGGAAAGGGUCACAGU	18	3455
  CCR5-4018	−	AUAAGGAAAGGGUCACAGU	19	3456
  CCR5-4019	−	GAUAAGGAAAGGGUCACAGU	20	3457
  CCR5-4020	−	AGAUAAGGAAAGGGUCACAGU	21	3458
  CCR5-4021	−	AAGAUAAGGAAAGGGUCACAGU	22	3459
  CCR5-4022	−	UAAGAUAAGGAAAGGGUCACAGU	23	3460
  CCR5-4023	−	UUAAGAUAAGGAAAGGGUCACAGU	24	3461
  CCR5-4024	−	AAAACAAAAUAAUCCAGU	18	3462
  CCR5-4025	−	AAAAACAAAAUAAUCCAGU	19	3463
  CCR5-4026	−	CAAAAACAAAAUAAUCCAGU	20	3464
  CCR5-4027	−	ACAAAAACAAAAUAAUCCAGU	21	3465
  CCR5-4028	−	AACAAAAACAAAAUAAUCCAGU	22	3466
  CCR5-4029	−	GAACAAAAACAAAAUAAUCCAGU	23	3467
  CCR5-4030	−	AGAACAAAAACAAAAUAAUCCAGU	24	3468
  CCR5-4031	−	UGGGUGGUGAGCAUCUGU	18	3469
  CCR5-4032	−	UUGGGUGGUGAGCAUCUGU	19	3470
  CCR5-4033	−	AUUGGGUGGUGAGCAUCUGU	20	3471
  CCR5-4034	−	UAUUGGGUGGUGAGCAUCUGU	21	3472
  CCR5-4035	−	AUAUUGGGUGGUGAGCAUCUGU	22	3473
  CCR5-4036	−	AAUAUUGGGUGGUGAGCAUCUGU	23	3474
  CCR5-4037	−	UAAUAUUGGGUGGUGAGCAUCUGU	24	3475
  CCR5-4038	−	UGGCCUGUUAGUUAGCUU	18	3476
  CCR5-4039	−	UUGGCCUGUUAGUUAGCUU	19	3477
  CCR5-4040	−	CUUGGCCUGUUAGUUAGCUU	20	3478
  CCR5-4041	−	GCUUGGCCUGUUAGUUAGCUU	21	3479
  CCR5-4042	−	UGCUUGGCCUGUUAGUUAGCUU	22	3480
  CCR5-4043	−	CUGCUUGGCCUGUUAGUUAGCUU	23	3481
  CCR5-4044	−	GCUGCUUGGCCUGUUAGUUAGCUU	24	3482

• Table 6E provides exemplary targeting domains for knocking down the CCR5 gene selected according to the fifth tier parameters. Within the additional 500 bp (e.g., upstream or downstream) of a transcription start site (TSS), e.g., extending to 1 kb upstream and downstream of a TSS and PAM is NNGRRV. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a S. aureus eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 6E
  5th Tier

  gRNA	DNA		Target Site
  Name	Strand	Targeting Domain	Length	SEQ ID NO
  CCR5-4208	+	UGGAGGAAAAAGAAAAAA	18	3651
  CCR5-4209	+	CUGGAGGAAAAAGAAAAAA	19	3652
  CCR5-4210	+	UCUGGAGGAAAAAGAAAAAA	20	3653
  CCR5-4211	+	GUCUGGAGGAAAAAGAAAAAA	21	3654
  CCR5-4212	+	UGUCUGGAGGAAAAAGAAAAAA	22	3655
  CCR5-4213	+	UUGUCUGGAGGAAAAAGAAAAAA	23	3656
  CCR5-4214	+	CUUGUCUGGAGGAAAAAGAAAAAA	24	3657
  CCR5-4215	+	UCUGGAGGAAAAAGAAAA	18	3658
  CCR5-4216	+	GUCUGGAGGAAAAAGAAAA	19	3659
  CCR5-4217	+	UGUCUGGAGGAAAAAGAAAA	20	3660
  CCR5-4218	+	UUGUCUGGAGGAAAAAGAAAA	21	3661
  CCR5-4219	+	CUUGUCUGGAGGAAAAAGAAAA	22	3662
  CCR5-4220	+	UCUUGUCUGGAGGAAAAAGAAAA	23	3663
  CCR5-4221	+	CUCUUGUCUGGAGGAAAAAGAAAA	24	3664
  CCR5-4222	+	CCUCUUGUCUGGAGGAAA	18	3665
  CCR5-4223	+	CCCUCUUGUCUGGAGGAAA	19	3666
  CCR5-4224	+	UCCCUCUUGUCUGGAGGAAA	20	3667
  CCR5-4225	+	UUCCCUCUUGUCUGGAGGAAA	21	3668
  CCR5-4226	+	CUUCCCUCUUGUCUGGAGGAAA	22	3669
  CCR5-4227	+	GCUUCCCUCUUGUCUGGAGGAAA	23	3670
  CCR5-4228	+	GGCUUCCCUCUUGUCUGGAGGAAA	24	3671
  CCR5-4229	+	GAUGUCACCAACCGCCAA	18	3672
  CCR5-4230	+	AGAUGUCACCAACCGCCAA	19	3673
  CCR5-4231	+	CAGAUGUCACCAACCGCCAA	20	3674
  CCR5-4232	+	UCAGAUGUCACCAACCGCCAA	21	3675
  CCR5-4233	+	UUCAGAUGUCACCAACCGCCAA	22	3676
  CCR5-4234	+	UUUCAGAUGUCACCAACCGCCAA	23	3677
  CCR5-4235	+	UUUUCAGAUGUCACCAACCGCCAA	24	3678
  CCR5-4236	+	CAAGGUCACGGAAGCCCA	18	3679
  CCR5-4237	+	CCAAGGUCACGGAAGCCCA	19	3680
  CCR5-4238	+	GCCAAGGUCACGGAAGCCCA	20	3681
  CCR5-4239	+	AGCCAAGGUCACGGAAGCCCA	21	3682
  CCR5-4240	+	GAGCCAAGGUCACGGAAGCCCA	22	3683
  CCR5-4241	+	AGAGCCAAGGUCACGGAAGCCCA	23	3684
  CCR5-4242	+	UAGAGCCAAGGUCACGGAAGCCCA	24	3685
  CCR5-4243	+	AUUCUAGAGCCAAGGUCA	18	3686
  CCR5-4244	+	UAUUCUAGAGCCAAGGUCA	19	3687
  CCR5-3069	+	UUAUUCUAGAGCCAAGGUCA	20	3688
  CCR5-4245	+	UUUAUUCUAGAGCCAAGGUCA	21	3689
  CCR5-4246	+	UUUUAUUCUAGAGCCAAGGUCA	22	3690
  CCR5-4247	+	UUUUUAUUCUAGAGCCAAGGUCA	23	3691
  CCR5-4248	+	CUUUUUAUUCUAGAGCCAAGGUCA	24	3692
  CCR5-4249	+	CCUGGGUCCAGAAAAAGA	18	3693
  CCR5-4250	+	UCCUGGGUCCAGAAAAAGA	19	3694
  CCR5-3071	+	AUCCUGGGUCCAGAAAAAGA	20	3695
  CCR5-4251	+	GAUCCUGGGUCCAGAAAAAGA	21	3696
  CCR5-4252	+	AGAUCCUGGGUCCAGAAAAAGA	22	3697
  CCR5-4253	+	AAGAUCCUGGGUCCAGAAAAAGA	23	3698
  CCR5-4254	+	UAAGAUCCUGGGUCCAGAAAAAGA	24	3699
  CCR5-4255	+	AACAAAAUAGUGAACAGA	18	3700
  CCR5-4256	+	CAACAAAAUAGUGAACAGA	19	3701
  CCR5-4257	+	GCAACAAAAUAGUGAACAGA	20	3702
  CCR5-4258	+	GGCAACAAAAUAGUGAACAGA	21	3703
  CCR5-4259	+	GGGCAACAAAAUAGUGAACAGA	22	3704
  CCR5-4260	+	AGGGCAACAAAAUAGUGAACAGA	23	3705
  CCR5-4261	+	AAGGGCAACAAAAUAGUGAACAGA	24	3706
  CCR5-4262	+	AGAUAGAUUAUAUCUGGA	18	3707
  CCR5-4263	+	CAGAUAGAUUAUAUCUGGA	19	3708
  CCR5-4264	+	UCAGAUAGAUUAUAUCUGGA	20	3709
  CCR5-4265	+	UUCAGAUAGAUUAUAUCUGGA	21	3710
  CCR5-4266	+	CUUCAGAUAGAUUAUAUCUGGA	22	3711
  CCR5-4267	+	GCUUCAGAUAGAUUAUAUCUGGA	23	3712
  CCR5-4268	+	AGCUUCAGAUAGAUUAUAUCUGGA	24	3713
  CCR5-4269	+	CUUAGACUAGGCAGCUGA	18	3714
  CCR5-4270	+	CCUUAGACUAGGCAGCUGA	19	3715
  CCR5-4271	+	ACCUUAGACUAGGCAGCUGA	20	3716
  CCR5-4272	+	CACCUUAGACUAGGCAGCUGA	21	3717
  CCR5-4273	+	GCACCUUAGACUAGGCAGCUGA	22	3718
  CCR5-4274	+	UGCACCUUAGACUAGGCAGCUGA	23	3719
  CCR5-4275	+	CUGCACCUUAGACUAGGCAGCUGA	24	3720
  CCR5-4276	+	UUGAAGGGCAACAAAAUA	18	3721
  CCR5-4277	+	UUUGAAGGGCAACAAAAUA	19	3722
  CCR5-4278	+	GUUUGAAGGGCAACAAAAUA	20	3723
  CCR5-4279	+	GGUUUGAAGGGCAACAAAAUA	21	3724
  CCR5-4280	+	UGGUUUGAAGGGCAACAAAAUA	22	3725
  CCR5-4281	+	CUGGUUUGAAGGGCAACAAAAUA	23	3726
  CCR5-4282	+	ACUGGUUUGAAGGGCAACAAAAUA	24	3727
  CCR5-4283	+	GUAUAUAGUAUAGUCAUA	18	3728
  CCR5-4284	+	UGUAUAUAGUAUAGUCAUA	19	3729
  CCR5-4285	+	CUGUAUAUAGUAUAGUCAUA	20	3730
  CCR5-4286	+	ACUGUAUAUAGUAUAGUCAUA	21	3731
  CCR5-4287	+	GACUGUAUAUAGUAUAGUCAUA	22	3732
  CCR5-4288	+	UGACUGUAUAUAGUAUAGUCAUA	23	3733
  CCR5-4289	+	AUGACUGUAUAUAGUAUAGUCAUA	24	3734
  CCR5-4290	+	CAUGAAACUGAUAUAUUA	18	3735
  CCR5-4291	+	CCAUGAAACUGAUAUAUUA	19	3736
  CCR5-4292	+	GCCAUGAAACUGAUAUAUUA	20	3737
  CCR5-4293	+	UGCCAUGAAACUGAUAUAUUA	21	3738
  CCR5-4294	+	GUGCCAUGAAACUGAUAUAUUA	22	3739
  CCR5-4295	+	UGUGCCAUGAAACUGAUAUAUUA	23	3740
  CCR5-4296	+	CUGUGCCAUGAAACUGAUAUAUUA	24	3741
  CCR5-4297	+	AGUAUAGUCAUAAAGAAC	18	3742
  CCR5-4298	+	UAGUAUAGUCAUAAAGAAC	19	3743
  CCR5-4299	+	AUAGUAUAGUCAUAAAGAAC	20	3744
  CCR5-4300	+	UAUAGUAUAGUCAUAAAGAAC	21	3745
  CCR5-4301	+	AUAUAGUAUAGUCAUAAAGAAC	22	3746
  CCR5-4302	+	UAUAUAGUAUAGUCAUAAAGAAC	23	3747
  CCR5-4303	+	GUAUAUAGUAUAGUCAUAAAGAAC	24	3748
  CCR5-4304	+	CAGCUCUGCUGACAAUAC	18	3749
  CCR5-4305	+	UCAGCUCUGCUGACAAUAC	19	3750
  CCR5-4306	+	CUCAGCUCUGCUGACAAUAC	20	3751
  CCR5-4307	+	UCUCAGCUCUGCUGACAAUAC	21	3752
  CCR5-4308	+	UUCUCAGCUCUGCUGACAAUAC	22	3753
  CCR5-4309	+	CUUCUCAGCUCUGCUGACAAUAC	23	3754
  CCR5-4310	+	UCUUCUCAGCUCUGCUGACAAUAC	24	3755
  CCR5-4311	+	AACCUGUUUAGCUCACCC	18	3756
  CCR5-4312	+	AAACCUGUUUAGCUCACCC	19	3757
  CCR5-4313	+	GAAACCUGUUUAGCUCACCC	20	3758
  CCR5-4314	+	GGAAACCUGUUUAGCUCACCC	21	3759
  CCR5-4315	+	GGGAAACCUGUUUAGCUCACCC	22	3760
  CCR5-4316	+	UGGGAAACCUGUUUAGCUCACCC	23	3761
  CCR5-4317	+	AUGGGAAACCUGUUUAGCUCACCC	24	3762
  CCR5-4318	+	GAGUUGUCAUACAUACCC	18	3763
  CCR5-4319	+	AGAGUUGUCAUACAUACCC	19	3764
  CCR5-4320	+	AAGAGUUGUCAUACAUACCC	20	3765
  CCR5-4321	+	UAAGAGUUGUCAUACAUACCC	21	3766
  CCR5-4322	+	UUAAGAGUUGUCAUACAUACCC	22	3767
  CCR5-4323	+	AUUAAGAGUUGUCAUACAUACCC	23	3768
  CCR5-4324	+	AAUUAAGAGUUGUCAUACAUACCC	24	3769
  CCR5-4325	+	GCAGCUGAGAGAAGCCCC	18	3770
  CCR5-4326	+	GGCAGCUGAGAGAAGCCCC	19	3771
  CCR5-4327	+	AGGCAGCUGAGAGAAGCCCC	20	3772
  CCR5-4328	+	UAGGCAGCUGAGAGAAGCCCC	21	3773
  CCR5-4329	+	CUAGGCAGCUGAGAGAAGCCCC	22	3774
  CCR5-4330	+	ACUAGGCAGCUGAGAGAAGCCCC	23	3775
  CCR5-4331	+	GACUAGGCAGCUGAGAGAAGCCCC	24	3776
  CCR5-4332	+	GCCAAGGUCACGGAAGCC	18	3777
  CCR5-4333	+	AGCCAAGGUCACGGAAGCC	19	3778
  CCR5-4334	+	GAGCCAAGGUCACGGAAGCC	20	3779
  CCR5-4335	+	AGAGCCAAGGUCACGGAAGCC	21	3780
  CCR5-4336	+	UAGAGCCAAGGUCACGGAAGCC	22	3781
  CCR5-4337	+	CUAGAGCCAAGGUCACGGAAGCC	23	3782
  CCR5-4338	+	UCUAGAGCCAAGGUCACGGAAGCC	24	3783
  CCR5-4339	+	CAGAUGUCACCAACCGCC	18	3784
  CCR5-4340	+	UCAGAUGUCACCAACCGCC	19	3785
  CCR5-4341	+	UUCAGAUGUCACCAACCGCC	20	3786
  CCR5-4342	+	UUUCAGAUGUCACCAACCGCC	21	3787
  CCR5-4343	+	UUUUCAGAUGUCACCAACCGCC	22	3788
  CCR5-4344	+	AUUUUCAGAUGUCACCAACCGCC	23	3789
  CCR5-4345	+	GAUUUUCAGAUGUCACCAACCGCC	24	3790
  CCR5-4346	+	UUAUAUACUAACUGUGCC	18	3791
  CCR5-4347	+	AUUAUAUACUAACUGUGCC	19	3792
  CCR5-4348	+	AAUUAUAUACUAACUGUGCC	20	3793
  CCR5-4349	+	GAAUUAUAUACUAACUGUGCC	21	3794
  CCR5-4350	+	AGAAUUAUAUACUAACUGUGCC	22	3795
  CCR5-4351	+	AAGAAUUAUAUACUAACUGUGCC	23	3796
  CCR5-4352	+	AAAGAAUUAUAUACUAACUGUGCC	24	3797
  CCR5-4353	+	CAGAGGGCAUCUUGUGGC	18	3798
  CCR5-4354	+	CCAGAGGGCAUCUUGUGGC	19	3799
  CCR5-4355	+	CCCAGAGGGCAUCUUGUGGC	20	3800
  CCR5-4356	+	GCCCAGAGGGCAUCUUGUGGC	21	3801
  CCR5-4357	+	AGCCCAGAGGGCAUCUUGUGGC	22	3802
  CCR5-4358	+	AAGCCCAGAGGGCAUCUUGUGGC	23	3803
  CCR5-4359	+	GAAGCCCAGAGGGCAUCUUGUGGC	24	3804
  CCR5-4360	+	UAUUCUAGAGCCAAGGUC	18	3805
  CCR5-4361	+	UUAUUCUAGAGCCAAGGUC	19	3806
  CCR5-4362	+	UUUAUUCUAGAGCCAAGGUC	20	3807
  CCR5-4363	+	UUUUAUUCUAGAGCCAAGGUC	21	3808
  CCR5-4364	+	UUUUUAUUCUAGAGCCAAGGUC	22	3809
  CCR5-4365	+	CUUUUUAUUCUAGAGCCAAGGUC	23	3810
  CCR5-4366	+	GCUUUUUAUUCUAGAGCCAAGGUC	24	3811
  CCR5-4367	+	CCACUAAGAUCCUGGGUC	18	3812
  CCR5-4368	+	CCCACUAAGAUCCUGGGUC	19	3813
  CCR5-4369	+	CCCCACUAAGAUCCUGGGUC	20	3814
  CCR5-4370	+	UCCCCACUAAGAUCCUGGGUC	21	3815
  CCR5-4371	+	AUCCCCACUAAGAUCCUGGGUC	22	3816
  CCR5-4372	+	AAUCCCCACUAAGAUCCUGGGUC	23	3817
  CCR5-4373	+	AAAUCCCCACUAAGAUCCUGGGUC	24	3818
  CCR5-4374	+	UUAGGCUUCCCUCUUGUC	18	3819
  CCR5-4375	+	UUUAGGCUUCCCUCUUGUC	19	3820
  CCR5-3097	+	UUUUAGGCUUCCCUCUUGUC	20	3821
  CCR5-4376	+	UUUUUAGGCUUCCCUCUUGUC	21	3822
  CCR5-4377	+	AUUUUUAGGCUUCCCUCUUGUC	22	3823
  CCR5-4378	+	CAUUUUUAGGCUUCCCUCUUGUC	23	3824
  CCR5-4379	+	CCAUUUUUAGGCUUCCCUCUUGUC	24	3825
  CCR5-4380	+	AGCCAAAGCUUUUUAUUC	18	3826
  CCR5-4381	+	AAGCCAAAGCUUUUUAUUC	19	3827
  CCR5-4382	+	CAAGCCAAAGCUUUUUAUUC	20	3828
  CCR5-4383	+	ACAAGCCAAAGCUUUUUAUUC	21	3829
  CCR5-4384	+	CACAAGCCAAAGCUUUUUAUUC	22	3830
  CCR5-4385	+	UCACAAGCCAAAGCUUUUUAUUC	23	3831
  CCR5-4386	+	AUCACAAGCCAAAGCUUUUUAUUC	24	3832
  CCR5-4387	+	UCCUGGGUCCAGAAAAAG	18	3833
  CCR5-4388	+	AUCCUGGGUCCAGAAAAAG	19	3834
  CCR5-4389	+	GAUCCUGGGUCCAGAAAAAG	20	3835
  CCR5-4390	+	AGAUCCUGGGUCCAGAAAAAG	21	3836
  CCR5-4391	+	AAGAUCCUGGGUCCAGAAAAAG	22	3837
  CCR5-4392	+	UAAGAUCCUGGGUCCAGAAAAAG	23	3838
  CCR5-4393	+	CUAAGAUCCUGGGUCCAGAAAAAG	24	3839
  CCR5-4394	+	GCACCUUAGACUAGGCAG	18	3840
  CCR5-4395	+	UGCACCUUAGACUAGGCAG	19	3841
  CCR5-4396	+	CUGCACCUUAGACUAGGCAG	20	3842
  CCR5-4397	+	CCUGCACCUUAGACUAGGCAG	21	3843
  CCR5-4398	+	CCCUGCACCUUAGACUAGGCAG	22	3844
  CCR5-4399	+	UCCCUGCACCUUAGACUAGGCAG	23	3845
  CCR5-4400	+	CUCCCUGCACCUUAGACUAGGCAG	24	3846
  CCR5-4401	+	UAAGUUCAGCUGCUCUAG	18	3847
  CCR5-4402	+	UUAAGUUCAGCUGCUCUAG	19	3848
  CCR5-4403	+	UUUAAGUUCAGCUGCUCUAG	20	3849
  CCR5-4404	+	AUUUAAGUUCAGCUGCUCUAG	21	3850
  CCR5-4405	+	UAUUUAAGUUCAGCUGCUCUAG	22	3851
  CCR5-4406	+	CUAUUUAAGUUCAGCUGCUCUAG	23	3852
  CCR5-4407	+	UCUAUUUAAGUUCAGCUGCUCUAG	24	3853
  CCR5-4408	+	AUGAAACUGAUAUAUUAG	18	3854
  CCR5-4409	+	CAUGAAACUGAUAUAUUAG	19	3855
  CCR5-3105	+	CCAUGAAACUGAUAUAUUAG	20	3856
  CCR5-4410	+	GCCAUGAAACUGAUAUAUUAG	21	3857
  CCR5-4411	+	UGCCAUGAAACUGAUAUAUUAG	22	3858
  CCR5-4412	+	GUGCCAUGAAACUGAUAUAUUAG	23	3859
  CCR5-4413	+	UGUGCCAUGAAACUGAUAUAUUAG	24	3860
  CCR5-4414	+	GGCUUCCCUCUUGUCUGG	18	3861
  CCR5-4415	+	AGGCUUCCCUCUUGUCUGG	19	3862
  CCR5-3108	+	UAGGCUUCCCUCUUGUCUGG	20	3863
  CCR5-4416	+	UUAGGCUUCCCUCUUGUCUGG	21	3864
  CCR5-4417	+	UUUAGGCUUCCCUCUUGUCUGG	22	3865
  CCR5-4418	+	UUUUAGGCUUCCCUCUUGUCUGG	23	3866
  CCR5-4419	+	UUUUUAGGCUUCCCUCUUGUCUGG	24	3867
  CCR5-4420	+	CCAUAUACUUAUGUCAUG	18	3868
  CCR5-4421	+	ACCAUAUACUUAUGUCAUG	19	3869
  CCR5-3111	+	GACCAUAUACUUAUGUCAUG	20	3870
  CCR5-4422	+	UGACCAUAUACUUAUGUCAUG	21	3871
  CCR5-4423	+	UUGACCAUAUACUUAUGUCAUG	22	3872
  CCR5-4424	+	CUUGACCAUAUACUUAUGUCAUG	23	3873
  CCR5-4425	+	ACUUGACCAUAUACUUAUGUCAUG	24	3874
  CCR5-4426	+	AGGCUUCCCUCUUGUCUG	18	3875
  CCR5-4427	+	UAGGCUUCCCUCUUGUCUG	19	3876
  CCR5-4428	+	UUAGGCUUCCCUCUUGUCUG	20	3877
  CCR5-4429	+	UUUAGGCUUCCCUCUUGUCUG	21	3878
  CCR5-4430	+	UUUUAGGCUUCCCUCUUGUCUG	22	3879
  CCR5-4431	+	UUUUUAGGCUUCCCUCUUGUCUG	23	3880
  CCR5-4432	+	AUUUUUAGGCUUCCCUCUUGUCUG	24	3881
  CCR5-4433	+	UAAAUGCUUACUGGUUUG	18	3882
  CCR5-4434	+	AUAAAUGCUUACUGGUUUG	19	3883
  CCR5-4435	+	CAUAAAUGCUUACUGGUUUG	20	3884
  CCR5-4436	+	UCAUAAAUGCUUACUGGUUUG	21	3885
  CCR5-4437	+	CUCAUAAAUGCUUACUGGUUUG	22	3886
  CCR5-4438	+	CCUCAUAAAUGCUUACUGGUUUG	23	3887
  CCR5-4439	+	UCCUCAUAAAUGCUUACUGGUUUG	24	3888
  CCR5-4440	+	ACCAUAUACUUAUGUCAU	18	3889
  CCR5-4441	+	GACCAUAUACUUAUGUCAU	19	3890
  CCR5-4442	+	UGACCAUAUACUUAUGUCAU	20	3891
  CCR5-4443	+	UUGACCAUAUACUUAUGUCAU	21	3892
  CCR5-4444	+	CUUGACCAUAUACUUAUGUCAU	22	3893
  CCR5-4445	+	ACUUGACCAUAUACUUAUGUCAU	23	3894
  CCR5-4446	+	AACUUGACCAUAUACUUAUGUCAU	24	3895
  CCR5-4447	+	CUGGGUCCAGAAAAAGAU	18	3896
  CCR5-4448	+	CCUGGGUCCAGAAAAAGAU	19	3897
  CCR5-3122	+	UCCUGGGUCCAGAAAAAGAU	20	3898
  CCR5-4449	+	AUCCUGGGUCCAGAAAAAGAU	21	3899
  CCR5-4450	+	GAUCCUGGGUCCAGAAAAAGAU	22	3900
  CCR5-4451	+	AGAUCCUGGGUCCAGAAAAAGAU	23	3901
  CCR5-4452	+	AAGAUCCUGGGUCCAGAAAAAGAU	24	3902
  CCR5-4453	+	GCCAUGAAACUGAUAUAU	18	3903
  CCR5-4454	+	UGCCAUGAAACUGAUAUAU	19	3904
  CCR5-4455	+	GUGCCAUGAAACUGAUAUAU	20	3905
  CCR5-4456	+	UGUGCCAUGAAACUGAUAUAU	21	3906
  CCR5-4457	+	CUGUGCCAUGAAACUGAUAUAU	22	3907
  CCR5-4458	+	ACUGUGCCAUGAAACUGAUAUAU	23	3908
  CCR5-4459	+	AACUGUGCCAUGAAACUGAUAUAU	24	3909
  CCR5-4460	+	GCUUCAGAUAGAUUAUAU	18	3910
  CCR5-4461	+	AGCUUCAGAUAGAUUAUAU	19	3911
  CCR5-4462	+	UAGCUUCAGAUAGAUUAUAU	20	3912
  CCR5-4463	+	AUAGCUUCAGAUAGAUUAUAU	21	3913
  CCR5-4464	+	CAUAGCUUCAGAUAGAUUAUAU	22	3914
  CCR5-4465	+	UCAUAGCUUCAGAUAGAUUAUAU	23	3915
  CCR5-4466	+	CUCAUAGCUUCAGAUAGAUUAUAU	24	3916
  CCR5-4467	+	ACCUUAGACUAGGCAGCU	18	3917
  CCR5-4468	+	CACCUUAGACUAGGCAGCU	19	3918
  CCR5-4469	+	GCACCUUAGACUAGGCAGCU	20	3919
  CCR5-4470	+	UGCACCUUAGACUAGGCAGCU	21	3920
  CCR5-4471	+	CUGCACCUUAGACUAGGCAGCU	22	3921
  CCR5-4472	+	CCUGCACCUUAGACUAGGCAGCU	23	3922
  CCR5-4473	+	CCCUGCACCUUAGACUAGGCAGCU	24	3923
  CCR5-4474	+	AGAGGGCAUCUUGUGGCU	18	3924
  CCR5-4475	+	CAGAGGGCAUCUUGUGGCU	19	3925
  CCR5-3129	+	CCAGAGGGCAUCUUGUGGCU	20	3926
  CCR5-4476	+	CCCAGAGGGCAUCUUGUGGCU	21	3927
  CCR5-4477	+	GCCCAGAGGGCAUCUUGUGGCU	22	3928
  CCR5-4478	+	AGCCCAGAGGGCAUCUUGUGGCU	23	3929
  CCR5-4479	+	AAGCCCAGAGGGCAUCUUGUGGCU	24	3930
  CCR5-4480	+	GGGUCUCAUUUGCCUUCU	18	3931
  CCR5-4481	+	GGGGUCUCAUUUGCCUUCU	19	3932
  CCR5-4482	+	UGGGGUCUCAUUUGCCUUCU	20	3933
  CCR5-4483	+	UUGGGGUCUCAUUUGCCUUCU	21	3934
  CCR5-4484	+	UUUGGGGUCUCAUUUGCCUUCU	22	3935
  CCR5-4485	+	GUUUGGGGUCUCAUUUGCCUUCU	23	3936
  CCR5-4486	+	UGUUUGGGGUCUCAUUUGCCUUCU	24	3937
  CCR5-4487	+	AAAAUCCUCACAUUUUCU	18	3938
  CCR5-4488	+	UAAAAUCCUCACAUUUUCU	19	3939
  CCR5-4489	+	GUAAAAUCCUCACAUUUUCU	20	3940
  CCR5-4490	+	UGUAAAAUCCUCACAUUUUCU	21	3941
  CCR5-4491	+	UUGUAAAAUCCUCACAUUUUCU	22	3942
  CCR5-4492	+	AUUGUAAAAUCCUCACAUUUUCU	23	3943
  CCR5-4493	+	AAUUGUAAAAUCCUCACAUUUUCU	24	3944
  CCR5-4494	+	UCAUAAAUGCUUACUGGU	18	3945
  CCR5-4495	+	CUCAUAAAUGCUUACUGGU	19	3946
  CCR5-4496	+	CCUCAUAAAUGCUUACUGGU	20	3947
  CCR5-4497	+	UCCUCAUAAAUGCUUACUGGU	21	3948
  CCR5-4498	+	GUCCUCAUAAAUGCUUACUGGU	22	3949
  CCR5-4499	+	AGUCCUCAUAAAUGCUUACUGGU	23	3950
  CCR5-4500	+	GAGUCCUCAUAAAUGCUUACUGGU	24	3951
  CCR5-4501	+	GGCACGUAAUUUUGCUGU	18	3952
  CCR5-4502	+	GGGCACGUAAUUUUGCUGU	19	3953
  CCR5-4503	+	GGGGCACGUAAUUUUGCUGU	20	3954
  CCR5-4504	+	GGGGGCACGUAAUUUUGCUGU	21	3955
  CCR5-4505	+	UGGGGGCACGUAAUUUUGCUGU	22	3956
  CCR5-4506	+	UUGGGGGCACGUAAUUUUGCUGU	23	3957
  CCR5-4507	+	AUUGGGGGCACGUAAUUUUGCUGU	24	3958
  CCR5-4508	+	UUUAGGCUUCCCUCUUGU	18	3959
  CCR5-4509	+	UUUUAGGCUUCCCUCUUGU	19	3960
  CCR5-4510	+	UUUUUAGGCUUCCCUCUUGU	20	3961
  CCR5-4511	+	AUUUUUAGGCUUCCCUCUUGU	21	3962
  CCR5-4512	+	CAUUUUUAGGCUUCCCUCUUGU	22	3963
  CCR5-4513	+	CCAUUUUUAGGCUUCCCUCUUGU	23	3964
  CCR5-4514	+	ACCAUUUUUAGGCUUCCCUCUUGU	24	3965
  CCR5-4515	+	AAAAGCUCAUUUUUAAUU	18	3966
  CCR5-4516	+	GAAAAGCUCAUUUUUAAUU	19	3967
  CCR5-4517	+	AGAAAAGCUCAUUUUUAAUU	20	3968
  CCR5-4518	+	UAGAAAAGCUCAUUUUUAAUU	21	3969
  CCR5-4519	+	CUAGAAAAGCUCAUUUUUAAUU	22	3970
  CCR5-4520	+	CCUAGAAAAGCUCAUUUUUAAUU	23	3971
  CCR5-4521	+	CCCUAGAAAAGCUCAUUUUUAAUU	24	3972
  CCR5-4522	+	ACUUAGACACAACUUCUU	18	3973
  CCR5-4523	+	GACUUAGACACAACUUCUU	19	3974
  CCR5-4524	+	AGACUUAGACACAACUUCUU	20	3975
  CCR5-4525	+	CAGACUUAGACACAACUUCUU	21	3976
  CCR5-4526	+	CCAGACUUAGACACAACUUCUU	22	3977
  CCR5-4527	+	ACCAGACUUAGACACAACUUCUU	23	3978
  CCR5-4528	+	AACCAGACUUAGACACAACUUCUU	24	3979
  CCR5-4529	−	UAUGGUUCAAAAUUAAAA	18	3980
  CCR5-4530	−	UUAUGGUUCAAAAUUAAAA	19	3981
  CCR5-4531	−	UUUAUGGUUCAAAAUUAAAA	20	3982
  CCR5-4532	−	CUUUAUGGUUCAAAAUUAAAA	21	3983
  CCR5-4533	−	UCUUUAUGGUUCAAAAUUAAAA	22	3984
  CCR5-4534	−	UUCUUUAUGGUUCAAAAUUAAAA	23	3985
  CCR5-4535	−	AUUCUUUAUGGUUCAAAAUUAAAA	24	3986
  CCR5-4536	−	UCUUUUUCCUCCAGACAA	18	3987
  CCR5-4537	−	UUCUUUUUCCUCCAGACAA	19	3988
  CCR5-4538	−	UUUCUUUUUCCUCCAGACAA	20	3989
  CCR5-4539	−	UUUUCUUUUUCCUCCAGACAA	21	3990
  CCR5-4540	−	UUUUUCUUUUUCCUCCAGACAA	22	3991
  CCR5-4541	−	UUUUUUCUUUUUCCUCCAGACAA	23	3992
  CCR5-4542	−	CUUUUUUCUUUUUCCUCCAGACAA	24	3993
  CCR5-4543	−	UGAUCUCUAAGAAGGCAA	18	3994
  CCR5-4544	−	GUGAUCUCUAAGAAGGCAA	19	3995
  CCR5-4545	−	UGUGAUCUCUAAGAAGGCAA	20	3996
  CCR5-4546	−	UUGUGAUCUCUAAGAAGGCAA	21	3997
  CCR5-4547	−	CUUGUGAUCUCUAAGAAGGCAA	22	3998
  CCR5-4548	−	GCUUGUGAUCUCUAAGAAGGCAA	23	3999
  CCR5-4549	−	GGCUUGUGAUCUCUAAGAAGGCAA	24	4000
  CCR5-4550	−	ACUCACAGGGUUUAAUAA	18	4001
  CCR5-4551	−	GACUCACAGGGUUUAAUAA	19	4002
  CCR5-4552	−	AGACUCACAGGGUUUAAUAA	20	4003
  CCR5-4553	−	GAGACUCACAGGGUUUAAUAA	21	4004
  CCR5-4554	−	UGAGACUCACAGGGUUUAAUAA	22	4005
  CCR5-4555	−	UUGAGACUCACAGGGUUUAAUAA	23	4006
  CCR5-4556	−	UUUGAGACUCACAGGGUUUAAUAA	24	4007
  CCR5-4557	−	AGAGCUGAGAAGACAGCA	18	4008
  CCR5-4558	−	CAGAGCUGAGAAGACAGCA	19	4009
  CCR5-4559	−	GCAGAGCUGAGAAGACAGCA	20	4010
  CCR5-4560	−	AGCAGAGCUGAGAAGACAGCA	21	4011
  CCR5-4561	−	CAGCAGAGCUGAGAAGACAGCA	22	4012
  CCR5-4562	−	UCAGCAGAGCUGAGAAGACAGCA	23	4013
  CCR5-4563	−	GUCAGCAGAGCUGAGAAGACAGCA	24	4014
  CCR5-4564	−	CUACAAACACAAACUUCA	18	4015
  CCR5-4565	−	ACUACAAACACAAACUUCA	19	4016
  CCR5-4566	−	AACUACAAACACAAACUUCA	20	4017
  CCR5-4567	−	AAACUACAAACACAAACUUCA	21	4018
  CCR5-4568	−	GAAACUACAAACACAAACUUCA	22	4019
  CCR5-4569	−	AGAAACUACAAACACAAACUUCA	23	4020
  CCR5-4570	−	CAGAAACUACAAACACAAACUUCA	24	4021
  CCR5-4571	−	UUUUUCCUCCAGACAAGA	18	4022
  CCR5-4572	−	CUUUUUCCUCCAGACAAGA	19	4023
  CCR5-3072	−	UCUUUUUCCUCCAGACAAGA	20	4024
  CCR5-4573	−	UUCUUUUUCCUCCAGACAAGA	21	4025
  CCR5-4574	−	UUUCUUUUUCCUCCAGACAAGA	22	4026
  CCR5-4575	−	UUUUCUUUUUCCUCCAGACAAGA	23	4027
  CCR5-4576	−	UUUUUCUUUUUCCUCCAGACAAGA	24	4028
  CCR5-4577	−	UACGUGCCCCCAAUCCUA	18	4029
  CCR5-4578	−	UUACGUGCCCCCAAUCCUA	19	4030
  CCR5-4579	−	AUUACGUGCCCCCAAUCCUA	20	4031
  CCR5-4580	−	AAUUACGUGCCCCCAAUCCUA	21	4032
  CCR5-4581	−	AAAUUACGUGCCCCCAAUCCUA	22	4033
  CCR5-4582	−	AAAAUUACGUGCCCCCAAUCCUA	23	4034
  CCR5-4583	−	CAAAAUUACGUGCCCCCAAUCCUA	24	4035
  CCR5-4584	−	UCUGGACCCAGGAUCUUA	18	4036
  CCR5-4585	−	UUCUGGACCCAGGAUCUUA	19	4037
  CCR5-4586	−	UUUCUGGACCCAGGAUCUUA	20	4038
  CCR5-4587	−	UUUUCUGGACCCAGGAUCUUA	21	4039
  CCR5-4588	−	UUUUUCUGGACCCAGGAUCUUA	22	4040
  CCR5-4589	−	CUUUUUCUGGACCCAGGAUCUUA	23	4041
  CCR5-4590	−	UCUUUUUCUGGACCCAGGAUCUUA	24	4042
  CCR5-4591	−	UUUCUUUUUCCUCCAGAC	18	4043
  CCR5-4592	−	UUUUCUUUUUCCUCCAGAC	19	4044
  CCR5-4593	−	UUUUUCUUUUUCCUCCAGAC	20	4045
  CCR5-4594	−	UUUUUUCUUUUUCCUCCAGAC	21	4046
  CCR5-4595	−	CUUUUUUCUUUUUCCUCCAGAC	22	4047
  CCR5-4596	−	UCUUUUUUCUUUUUCCUCCAGAC	23	4048
  CCR5-4597	−	CUCUUUUUUCUUUUUCCUCCAGAC	24	4049
  CCR5-4598	−	GUCAUCUAUGACCUUCCC	18	4050
  CCR5-4599	−	UGUCAUCUAUGACCUUCCC	19	4051
  CCR5-3087	−	UUGUCAUCUAUGACCUUCCC	20	4052
  CCR5-4600	−	GUUGUCAUCUAUGACCUUCCC	21	4053
  CCR5-4601	−	UGUUGUCAUCUAUGACCUUCCC	22	4054
  CCR5-4602	−	CUGUUGUCAUCUAUGACCUUCCC	23	4055
  CCR5-4603	−	GCUGUUGUCAUCUAUGACCUUCCC	24	4056
  CCR5-4604	−	UGUCAUCUAUGACCUUCC	18	4057
  CCR5-4605	−	UUGUCAUCUAUGACCUUCC	19	4058
  CCR5-4606	−	GUUGUCAUCUAUGACCUUCC	20	4059
  CCR5-4607	−	UGUUGUCAUCUAUGACCUUCC	21	4060
  CCR5-4608	−	CUGUUGUCAUCUAUGACCUUCC	22	4061
  CCR5-4609	−	GCUGUUGUCAUCUAUGACCUUCC	23	4062
  CCR5-4610	−	GGCUGUUGUCAUCUAUGACCUUCC	24	4063
  CCR5-4611	−	UAAGAGAAAAUUCUCAGC	18	4064
  CCR5-4612	−	AUAAGAGAAAAUUCUCAGC	19	4065
  CCR5-4613	−	AAUAAGAGAAAAUUCUCAGC	20	4066
  CCR5-4614	−	UAAUAAGAGAAAAUUCUCAGC	21	4067
  CCR5-4615	−	UUAAUAAGAGAAAAUUCUCAGC	22	4068
  CCR5-4616	−	UUUAAUAAGAGAAAAUUCUCAGC	23	4069
  CCR5-4617	−	GUUUAAUAAGAGAAAAUUCUCAGC	24	4070
  CCR5-4618	−	CUGCCUAGUCUAAGGUGC	18	4071
  CCR5-4619	−	GCUGCCUAGUCUAAGGUGC	19	4072
  CCR5-3091	−	AGCUGCCUAGUCUAAGGUGC	20	4073
  CCR5-4620	−	CAGCUGCCUAGUCUAAGGUGC	21	4074
  CCR5-4621	−	UCAGCUGCCUAGUCUAAGGUGC	22	4075
  CCR5-4622	−	CUCAGCUGCCUAGUCUAAGGUGC	23	4076
  CCR5-4623	−	UCUCAGCUGCCUAGUCUAAGGUGC	24	4077
  CCR5-4624	−	GACAGCAGAGAGCUACUC	18	4078
  CCR5-4625	−	AGACAGCAGAGAGCUACUC	19	4079
  CCR5-4626	−	AAGACAGCAGAGAGCUACUC	20	4080
  CCR5-4627	−	GAAGACAGCAGAGAGCUACUC	21	4081
  CCR5-4628	−	AGAAGACAGCAGAGAGCUACUC	22	4082
  CCR5-4629	−	GAGAAGACAGCAGAGAGCUACUC	23	4083
  CCR5-4630	−	UGAGAAGACAGCAGAGAGCUACUC	24	4084
  CCR5-4631	−	AUUAAAAAUGAGCUUUUC	18	4085
  CCR5-4632	−	AAUUAAAAAUGAGCUUUUC	19	4086
  CCR5-4633	−	AAAUUAAAAAUGAGCUUUUC	20	4087
  CCR5-4634	−	AAAAUUAAAAAUGAGCUUUUC	21	4088
  CCR5-4635	−	CAAAAUUAAAAAUGAGCUUUUC	22	4089
  CCR5-4636	−	UCAAAAUUAAAAAUGAGCUUUUC	23	4090
  CCR5-4637	−	UUCAAAAUUAAAAAUGAGCUUUUC	24	4091
  CCR5-4638	−	CUUUUUCCUCCAGACAAG	18	4092
  CCR5-4639	−	UCUUUUUCCUCCAGACAAG	19	4093
  CCR5-3101	−	UUCUUUUUCCUCCAGACAAG	20	4094
  CCR5-4640	−	UUUCUUUUUCCUCCAGACAAG	21	4095
  CCR5-4641	−	UUUUCUUUUUCCUCCAGACAAG	22	4096
  CCR5-4642	−	UUUUUCUUUUUCCUCCAGACAAG	23	4097
  CCR5-4643	−	UUUUUUCUUUUUCCUCCAGACAAG	24	4098
  CCR5-4644	−	GCAGAGCUGAGAAGACAG	18	4099
  CCR5-4645	−	AGCAGAGCUGAGAAGACAG	19	4100
  CCR5-4646	−	CAGCAGAGCUGAGAAGACAG	20	4101
  CCR5-4647	−	UCAGCAGAGCUGAGAAGACAG	21	4102
  CCR5-4648	−	GUCAGCAGAGCUGAGAAGACAG	22	4103
  CCR5-4649	−	UGUCAGCAGAGCUGAGAAGACAG	23	4104
  CCR5-4650	−	UUGUCAGCAGAGCUGAGAAGACAG	24	4105
  CCR5-4651	−	AAUUCUCAGCUAGAGCAG	18	4106
  CCR5-4652	−	AAAUUCUCAGCUAGAGCAG	19	4107
  CCR5-4653	−	AAAAUUCUCAGCUAGAGCAG	20	4108
  CCR5-4654	−	GAAAAUUCUCAGCUAGAGCAG	21	4109
  CCR5-4655	−	AGAAAAUUCUCAGCUAGAGCAG	22	4110
  CCR5-4656	−	GAGAAAAUUCUCAGCUAGAGCAG	23	4111
  CCR5-4657	−	AGAGAAAAUUCUCAGCUAGAGCAG	24	4112
  CCR5-4658	−	AUUCAUCUGUGGUGGCAG	18	4113
  CCR5-4659	−	CAUUCAUCUGUGGUGGCAG	19	4114
  CCR5-4660	−	ACAUUCAUCUGUGGUGGCAG	20	4115
  CCR5-4661	−	GACAUUCAUCUGUGGUGGCAG	21	4116
  CCR5-4662	−	UGACAUUCAUCUGUGGUGGCAG	22	4117
  CCR5-4663	−	AUGACAUUCAUCUGUGGUGGCAG	23	4118
  CCR5-4664	−	CAUGACAUUCAUCUGUGGUGGCAG	24	4119
  CCR5-4665	−	AAUCUCAAGUAUUGUCAG	18	4120
  CCR5-4666	−	AAAUCUCAAGUAUUGUCAG	19	4121
  CCR5-4667	−	AAAAUCUCAAGUAUUGUCAG	20	4122
  CCR5-4668	−	GAAAAUCUCAAGUAUUGUCAG	21	4123
  CCR5-4669	−	UGAAAAUCUCAAGUAUUGUCAG	22	4124
  CCR5-4670	−	CUGAAAAUCUCAAGUAUUGUCAG	23	4125
  CCR5-4671	−	UCUGAAAAUCUCAAGUAUUGUCAG	24	4126
  CCR5-4672	−	CAAGUAUUGUCAGCAGAG	18	4127
  CCR5-4673	−	UCAAGUAUUGUCAGCAGAG	19	4128
  CCR5-4674	−	CUCAAGUAUUGUCAGCAGAG	20	4129
  CCR5-4675	−	UCUCAAGUAUUGUCAGCAGAG	21	4130
  CCR5-4676	−	AUCUCAAGUAUUGUCAGCAGAG	22	4131
  CCR5-4677	−	AAUCUCAAGUAUUGUCAGCAGAG	23	4132
  CCR5-4678	−	AAAUCUCAAGUAUUGUCAGCAGAG	24	4133
  CCR5-4679	−	CUGGACCCAGGAUCUUAG	18	4134
  CCR5-4680	−	UCUGGACCCAGGAUCUUAG	19	4135
  CCR5-3106	−	UUCUGGACCCAGGAUCUUAG	20	4136
  CCR5-4681	−	UUUCUGGACCCAGGAUCUUAG	21	4137
  CCR5-4682	−	UUUUCUGGACCCAGGAUCUUAG	22	4138
  CCR5-4683	−	UUUUUCUGGACCCAGGAUCUUAG	23	4139
  CCR5-4684	−	CUUUUUCUGGACCCAGGAUCUUAG	24	4140
  CCR5-4685	−	UUAACUAUGGGCUCACGG	18	4141
  CCR5-4686	−	UUUAACUAUGGGCUCACGG	19	4142
  CCR5-4687	−	UUUUAACUAUGGGCUCACGG	20	4143
  CCR5-4688	−	GUUUUAACUAUGGGCUCACGG	21	4144
  CCR5-4689	−	AGUUUUAACUAUGGGCUCACGG	22	4145
  CCR5-4690	−	GAGUUUUAACUAUGGGCUCACGG	23	4146
  CCR5-4691	−	AGAGUUUUAACUAUGGGCUCACGG	24	4147
  CCR5-4692	−	GCUGCCUAGUCUAAGGUG	18	4148
  CCR5-4693	−	AGCUGCCUAGUCUAAGGUG	19	4149
  CCR5-4694	−	CAGCUGCCUAGUCUAAGGUG	20	4150
  CCR5-4695	−	UCAGCUGCCUAGUCUAAGGUG	21	4151
  CCR5-4696	−	CUCAGCUGCCUAGUCUAAGGUG	22	4152
  CCR5-4697	−	UCUCAGCUGCCUAGUCUAAGGUG	23	4153
  CCR5-4698	−	CUCUCAGCUGCCUAGUCUAAGGUG	24	4154
  CCR5-4699	−	ACAAACUUCACAGAAAAU	18	4155
  CCR5-4700	−	CACAAACUUCACAGAAAAU	19	4156
  CCR5-4701	−	ACACAAACUUCACAGAAAAU	20	4157
  CCR5-4702	−	AACACAAACUUCACAGAAAAU	21	4158
  CCR5-4703	−	AAACACAAACUUCACAGAAAAU	22	4159
  CCR5-4704	−	CAAACACAAACUUCACAGAAAAU	23	4160
  CCR5-4705	−	ACAAACACAAACUUCACAGAAAAU	24	4161
  CCR5-4706	−	AGACUCACAGGGUUUAAU	18	4162
  CCR5-4707	−	GAGACUCACAGGGUUUAAU	19	4163
  CCR5-4708	−	UGAGACUCACAGGGUUUAAU	20	4164
  CCR5-4709	−	UUGAGACUCACAGGGUUUAAU	21	4165
  CCR5-4710	−	UUUGAGACUCACAGGGUUUAAU	22	4166
  CCR5-4711	−	GUUUGAGACUCACAGGGUUUAAU	23	4167
  CCR5-4712	−	AGUUUGAGACUCACAGGGUUUAAU	24	4168
  CCR5-4713	−	CUUGGCGGUUGGUGACAU	18	4169
  CCR5-4714	−	UCUUGGCGGUUGGUGACAU	19	4170
  CCR5-4715	−	CUCUUGGCGGUUGGUGACAU	20	4171
  CCR5-4716	−	UCUCUUGGCGGUUGGUGACAU	21	4172
  CCR5-4717	−	CUCUCUUGGCGGUUGGUGACAU	22	4173
  CCR5-4718	−	GCUCUCUUGGCGGUUGGUGACAU	23	4174
  CCR5-4719	−	AGCUCUCUUGGCGGUUGGUGACAU	24	4175
  CCR5-4720	−	UAAUCUAUCUGAAGCUAU	18	4176
  CCR5-4721	−	AUAAUCUAUCUGAAGCUAU	19	4177
  CCR5-4722	−	UAUAAUCUAUCUGAAGCUAU	20	4178
  CCR5-4723	−	AUAUAAUCUAUCUGAAGCUAU	21	4179
  CCR5-4724	−	GAUAUAAUCUAUCUGAAGCUAU	22	4180
  CCR5-4725	−	AGAUAUAAUCUAUCUGAAGCUAU	23	4181
  CCR5-4726	−	CAGAUAUAAUCUAUCUGAAGCUAU	24	4182
  CCR5-4727	−	ACUCCAGAUAUAAUCUAU	18	4183
  CCR5-4728	−	CACUCCAGAUAUAAUCUAU	19	4184
  CCR5-4729	−	UCACUCCAGAUAUAAUCUAU	20	4185
  CCR5-4730	−	UUCACUCCAGAUAUAAUCUAU	21	4186
  CCR5-4731	−	CUUCACUCCAGAUAUAAUCUAU	22	4187
  CCR5-4732	−	UCUUCACUCCAGAUAUAAUCUAU	23	4188
  CCR5-4733	−	UUCUUCACUCCAGAUAUAAUCUAU	24	4189
  CCR5-4734	−	AAACCAGUAAGCAUUUAU	18	4190
  CCR5-4735	−	CAAACCAGUAAGCAUUUAU	19	4191
  CCR5-4736	−	UCAAACCAGUAAGCAUUUAU	20	4192
  CCR5-4737	−	UUCAAACCAGUAAGCAUUUAU	21	4193
  CCR5-4738	−	CUUCAAACCAGUAAGCAUUUAU	22	4194
  CCR5-4739	−	CCUUCAAACCAGUAAGCAUUUAU	23	4195
  CCR5-4740	−	CCCUUCAAACCAGUAAGCAUUUAU	24	4196
  CCR5-4741	−	CUCUUAAUUGUGGCAACU	18	4197
  CCR5-4742	−	ACUCUUAAUUGUGGCAACU	19	4198
  CCR5-4743	−	AACUCUUAAUUGUGGCAACU	20	4199
  CCR5-4744	−	CAACUCUUAAUUGUGGCAACU	21	4200
  CCR5-4745	−	ACAACUCUUAAUUGUGGCAACU	22	4201
  CCR5-4746	−	GACAACUCUUAAUUGUGGCAACU	23	4202
  CCR5-4747	−	UGACAACUCUUAAUUGUGGCAACU	24	4203
  CCR5-4748	−	GUCUAAAGAGUUUUAACU	18	4204
  CCR5-4749	−	UGUCUAAAGAGUUUUAACU	19	4205
  CCR5-4750	−	UUGUCUAAAGAGUUUUAACU	20	4206
  CCR5-4751	−	GUUGUCUAAAGAGUUUUAACU	21	4207
  CCR5-4752	−	UGUUGUCUAAAGAGUUUUAACU	22	4208
  CCR5-4753	−	CUGUUGUCUAAAGAGUUUUAACU	23	4209
  CCR5-4754	−	CCUGUUGUCUAAAGAGUUUUAACU	24	4210
  CCR5-4755	−	CGAGCCACAAGAUGCCCU	18	4211
  CCR5-4756	−	CCGAGCCACAAGAUGCCCU	19	4212
  CCR5-4757	−	CCCGAGCCACAAGAUGCCCU	20	4213
  CCR5-4758	−	UCCCGAGCCACAAGAUGCCCU	21	4214
  CCR5-4759	−	CUCCCGAGCCACAAGAUGCCCU	22	4215
  CCR5-4760	−	ACUCCCGAGCCACAAGAUGCCCU	23	4216
  CCR5-4761	−	UACUCCCGAGCCACAAGAUGCCCU	24	4217
  CCR5-4762	−	UAUAAUCUAUCUGAAGCU	18	4218
  CCR5-4763	−	AUAUAAUCUAUCUGAAGCU	19	4219
  CCR5-4764	−	GAUAUAAUCUAUCUGAAGCU	20	4220
  CCR5-4765	−	AGAUAUAAUCUAUCUGAAGCU	21	4221
  CCR5-4766	−	CAGAUAUAAUCUAUCUGAAGCU	22	4222
  CCR5-4767	−	CCAGAUAUAAUCUAUCUGAAGCU	23	4223
  CCR5-4768	−	UCCAGAUAUAAUCUAUCUGAAGCU	24	4224
  CCR5-4769	−	AGUAUUGUCAGCAGAGCU	18	4225
  CCR5-4770	−	AAGUAUUGUCAGCAGAGCU	19	4226
  CCR5-4771	−	CAAGUAUUGUCAGCAGAGCU	20	4227
  CCR5-4772	−	UCAAGUAUUGUCAGCAGAGCU	21	4228
  CCR5-4773	−	CUCAAGUAUUGUCAGCAGAGCU	22	4229
  CCR5-4774	−	UCUCAAGUAUUGUCAGCAGAGCU	23	4230
  CCR5-4775	−	AUCUCAAGUAUUGUCAGCAGAGCU	24	4231
  CCR5-4776	−	CUUUGGCUUGUGAUCUCU	18	4232
  CCR5-4777	−	GCUUUGGCUUGUGAUCUCU	19	4233
  CCR5-4778	−	AGCUUUGGCUUGUGAUCUCU	20	4234
  CCR5-4779	−	AAGCUUUGGCUUGUGAUCUCU	21	4235
  CCR5-4780	−	AAAGCUUUGGCUUGUGAUCUCU	22	4236
  CCR5-4781	−	AAAAGCUUUGGCUUGUGAUCUCU	23	4237
  CCR5-4782	−	AAAAAGCUUUGGCUUGUGAUCUCU	24	4238
  CCR5-4783	−	UUAAAAAUGAGCUUUUCU	18	4239
  CCR5-4784	−	AUUAAAAAUGAGCUUUUCU	19	4240
  CCR5-3134	−	AAUUAAAAAUGAGCUUUUCU	20	4241
  CCR5-4785	−	AAAUUAAAAAUGAGCUUUUCU	21	4242
  CCR5-4786	−	AAAAUUAAAAAUGAGCUUUUCU	22	4243
  CCR5-4787	−	CAAAAUUAAAAAUGAGCUUUUCU	23	4244
  CCR5-4788	−	UCAAAAUUAAAAAUGAGCUUUUCU	24	4245
  CCR5-4789	−	GUCUAAGGUGCAGGGAGU	18	4246
  CCR5-4790	−	AGUCUAAGGUGCAGGGAGU	19	4247
  CCR5-4791	−	UAGUCUAAGGUGCAGGGAGU	20	4248
  CCR5-4792	−	CUAGUCUAAGGUGCAGGGAGU	21	4249
  CCR5-4793	−	CCUAGUCUAAGGUGCAGGGAGU	22	4250
  CCR5-4794	−	GCCUAGUCUAAGGUGCAGGGAGU	23	4251
  CCR5-4795	−	UGCCUAGUCUAAGGUGCAGGGAGU	24	4252
  CCR5-4796	−	UCAAACCAGUAAGCAUUU	18	4253
  CCR5-4797	−	UUCAAACCAGUAAGCAUUU	19	4254
  CCR5-4798	−	CUUCAAACCAGUAAGCAUUU	20	4255
  CCR5-4799	−	CCUUCAAACCAGUAAGCAUUU	21	4256
  CCR5-4800	−	CCCUUCAAACCAGUAAGCAUUU	22	4257
  CCR5-4801	−	GCCCUUCAAACCAGUAAGCAUUU	23	4258
  CCR5-4802	−	UGCCCUUCAAACCAGUAAGCAUUU	24	4259
  CCR5-4803	−	CAGGUUUCCCAUCUUUUU	18	4260
  CCR5-4804	−	ACAGGUUUCCCAUCUUUUU	19	4261
  CCR5-4805	−	AACAGGUUUCCCAUCUUUUU	20	4262
  CCR5-4806	−	AAACAGGUUUCCCAUCUUUUU	21	4263
  CCR5-4807	−	UAAACAGGUUUCCCAUCUUUUU	22	4264
  CCR5-4808	−	CUAAACAGGUUUCCCAUCUUUUU	23	4265
  CCR5-4809	−	GCUAAACAGGUUUCCCAUCUUUUU	24	4266

• Table 7A provides exemplary targeting domains for knocking down the CCR5 gene selected according to the first tier parameters. The targeting domains bind within 500 bp (e.g., upstream or downstream) of a transcription start site (TSS) and have a high level of orthogonality. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 7A
  1st Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-4810	−	AUCCUUACCUCUCAAAA	17	4267
  CCR5-4811	+	CUAAAAGGUUAAGAAAA	17	4268
  CCR5-4812	−	AGCUGCUUGGCCUGUUA	17	4269
  CCR5-4813	+	AUUACUAUCCAAGAAGC	17	4270
  CCR5-4814	−	GUGAUCUUGUACAAAUC	17	4271
  CCR5-4815	−	CCGGUAAGUAACCUCUC	17	4272
  CCR5-4816	+	AUUUACGGGCUUUUCUC	17	4273
  CCR5-4817	−	AGACCAGAGAUCUAUUC	17	4274
  CCR5-4818	+	GUUCUCCUUAGCAGAAG	17	4275
  CCR5-4819	+	AUCUUUCUUUUGAGAGG	17	4276
  CCR5-4820	−	UUUUAUACUGUCUAUAU	17	4277
  CCR5-4821	−	UUCGCCUUCAAUACACU	17	4278
  CCR5-4822	+	UGACCCUUUCCUUAUCU	17	4279
  CCR5-4823	−	CUACUUUUAUACUGUCU	17	4280
  CCR5-4824	−	UAAAAAGAAGAACUGUU	17	4281
  CCR5-4825	+	GGUCUGAAGGUUUAUUU	17	4282
  CCR5-4826	−	ACAAUCCUUACCUCUCAAAA	20	4283
  CCR5-4827	+	AGGCUAAAAGGUUAAGAAAA	20	4284
  CCR5-4828	−	UACAUUUAAAGUUGGUUUAA	20	4285
  CCR5-4829	−	CUCAGCUGCUUGGCCUGUUA	20	4286
  CCR5-4830	+	GAAAUUACUAUCCAAGAAGC	20	4287
  CCR5-4831	−	CCUGUGAUCUUGUACAAAUC	20	4288
  CCR5-4832	−	UCCCCGGUAAGUAACCUCUC	20	4289
  CCR5-4833	+	UUUAUUUACGGGCUUUUCUC	20	4290
  CCR5-4834	−	UUCAGACCAGAGAUCUAUUC	20	4291
  CCR5-4835	+	UUAGUUCUCCUUAGCAGAAG	20	4292
  CCR5-3491	+	GAACAGUUCUUCUUUUUAAG	20	4293
  CCR5-4836	+	CAAAUCUUUCUUUUGAGAGG	20	4294
  CCR5-4837	−	UACUUUUAUACUGUCUAUAU	20	4295
  CCR5-4838	−	CUUUUCGCCUUCAAUACACU	20	4296
  CCR5-4839	+	CUGUGACCCUUUCCUUAUCU	20	4297
  CCR5-4840	−	UUCCUACUUUUAUACUGUCU	20	4298
  CCR5-4841	+	CCUUAGCAGAAGAUAAGAUU	20	4299
  CCR5-4842	−	ACUUAAAAAGAAGAACUGUU	20	4300
  CCR5-3668	+	UCUGGUCUGAAGGUUUAUUU	20	4301

• Table 7B provides exemplary targeting domains for knocking down the CCR5 gene selected according to the second tier parameters. The targeting domains bind within 500 bp (e.g., upstream or downstream) of a transcription start site (TSS). It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 7B
  2nd Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-4843	−	AACAUCAAAGAUACAAA	17	4302
  CCR5-4844	−	AUUUAAAGUUGGUUUAA	17	4303
  CCR5-4845	+	UGAUUUGUACAAGAUCA	17	4304
  CCR5-4846	+	CAGUUCUUCUUUUUAAG	17	4305
  CCR5-4847	−	AUUUCUUUUACUAAAAU	17	4306
  CCR5-4848	−	UAUUCUUUAUAUUUUCU	17	4307
  CCR5-4849	+	UAGCAGAAGAUAAGAUU	17	4308
  CCR5-4850	−	UAUAACAUCAAAGAUACAAA	20	4309
  CCR5-3386	+	AAAUGAUUUGUACAAGAUCA	20	4310
  CCR5-3978	−	GUAAUUUCUUUUACUAAAAU	20	4311
  CCR5-4851	−	CUUUAUUCUUUAUAUUUUCU	20	4312

• Table 7C provides exemplary targeting domains for knocking down the CCR5 gene selected according to the third tier parameters. Within the additional 500 bp (e.g., upstream or downstream) of a transcription start site (TSS), e.g., extending to 1 kb upstream and downstream of a TSS. It is contemplated herein that in an embodiment the targeting domain hybridizes to the target domain through complementary base pairing. Any of the targeting domains in the table can be used with a N. meningitidis eiCas9 molecule or eiCas9 fusion protein (e.g., an eiCas9 fused to a transcription repressor domain) to alter the CCR5 gene (e.g., reduce or eliminate CCR5 gene expression, CCR5 protein function, or the level of CCR5 protein). One or more gRNAs may be used to target an eiCas9 to the promoter region of the CCR5 gene.

• TABLE 7C
  3rd Tier

  			Target
  gRNA	DNA		Site	SEQ ID
  Name	Strand	Targeting Domain	Length	NO
  CCR5-4852	−	AUGGUUCAAAAUUAAAA	17	4313
  CCR5-4853	+	AUGUCACCAACCGCCAA	17	4314
  CCR5-4854	+	AAUUUCUCAUAGCUUCA	17	4315
  CCR5-4855	−	ACCUUGGCUCUAGAAUA	17	4316
  CCR5-4856	+	AGCUCUGCUGACAAUAC	17	4317
  CCR5-4857	−	GCUCUAGAAUAAAAAGC	17	4318
  CCR5-4858	+	UCUUAGAGAUCACAAGC	17	4319
  CCR5-3022	−	UGGACCCAGGAUCUUAG	17	4320
  CCR5-4859	−	AAACUUCACAGAAAAUG	17	4321
  CCR5-4860	−	UGCCAGAUACAUAGGUG	17	4322
  CCR5-4861	+	AUAGUGUGAGUCCUCAU	17	4323
  CCR5-4862	−	GAGCCACAAGAUGCCCU	17	4324
  CCR5-4863	+	UCAUGUGGAAAAUUUCU	17	4325
  CCR5-3052	−	UAAAAAUGAGCUUUUCU	17	4326
  CCR5-4864	+	AUUAAUUUUGACCAUUU	17	4327
  CCR5-4531	−	UUUAUGGUUCAAAAUUAAAA	20	4328
  CCR5-4231	+	CAGAUGUCACCAACCGCCAA	20	4329
  CCR5-4865	+	GAAAAUUUCUCAUAGCUUCA	20	4330
  CCR5-4866	−	GUGACCUUGGCUCUAGAAUA	20	4331
  CCR5-4306	+	CUCAGCUCUGCUGACAAUAC	20	4332
  CCR5-4867	−	UUGGCUCUAGAAUAAAAAGC	20	4333
  CCR5-4868	+	CCUUCUUAGAGAUCACAAGC	20	4334
  CCR5-3106	−	UUCUGGACCCAGGAUCUUAG	20	4335
  CCR5-4869	−	CACAAACUUCACAGAAAAUG	20	4336
  CCR5-4870	−	CUAUGCCAGAUACAUAGGUG	20	4337
  CCR5-4871	+	GGCAUAGUGUGAGUCCUCAU	20	4338
  CCR5-4757	−	CCCGAGCCACAAGAUGCCCU	20	4339
  CCR5-4872	+	AUGUCAUGUGGAAAAUUUCU	20	4340
  CCR5-3134	−	AAUUAAAAAUGAGCUUUUCU	20	4341
  CCR5-4873	+	AAUAUUAAUUUUGACCAUUU	20	4342

III. Cas9 Molecules
• Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes, S. aureus, and S. thermophilus Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and S. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them, e.g., Staphylococcus aureus and Neisseria meningitides Cas9 molecules. Additional Cas9 species include: Acidovorax avenae, Actinobacillus pleuropneumonias, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae.
• A Cas9 molecule, or Cas9 polypeptide, as that term is used herein, refers to a molecule or polypeptide that can interact with a guide RNA (gRNA) molecule and, in concert with the gRNA molecule, home or localizes to a site which comprises a target domain and PAM sequence. Cas9 molecule and Cas9 polypeptide, as those terms are used herein, refer to naturally occurring Cas9 molecules and to engineered, altered, or modified Cas9 molecules or Cas9 polypeptides that differ, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule or a sequence of Table 8.
• Cas9 Domains
• Crystal structures have been determined for two different naturally occurring bacterial Cas9 molecules (Jinek et al., Science, 343(6176):1247997, 2014) and for S. pyogenes Cas9 with a guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) (Nishimasu et al., Cell, 156:935-949, 2014; and Anders et al., Nature, 2014, doi: 10.1038/nature13579).
• A naturally occurring Cas9 molecule comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which further comprises domains described herein. FIGS. 9A-9B provide a schematic of the organization of important Cas9 domains in the primary structure. The domain nomenclature and the numbering of the amino acid residues encompassed by each domain used throughout this disclosure is as described in Nishimasu et al. The numbering of the amino acid residues is with reference to Cas9 from S. pyogenes.
• The REC lobe comprises the arginine-rich bridge helix (BH), the REC1 domain, and the REC2 domain. The REC lobe does not share structural similarity with other known proteins, indicating that it is a Cas9-specific functional domain. The BH domain is a long a helix and arginine rich region and comprises amino acids 60-93 of the sequence of S. pyogenes Cas9. The REC1 domain is important for recognition of the repeat:anti-repeat duplex, e.g., of a gRNA or a tracrRNA, and is therefore critical for Cas9 activity by recognizing the target sequence. The REC1 domain comprises two REC1 motifs at amino acids 94 to 179 and 308 to 717 of the sequence of S. pyogenes Cas9. These two REC1 domains, though separated by the REC2 domain in the linear primary structure, assemble in the tertiary structure to form the REC1 domain. The REC2 domain, or parts thereof, may also play a role in the recognition of the repeat:anti-repeat duplex. The REC2 domain comprises amino acids 180-307 of the sequence of S. pyogenes Cas9.
• The NUC lobe comprises the RuvC domain (also referred to herein as RuvC-like domain), the HNH domain (also referred to herein as HNH-like domain), and the PAM-interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves a single strand, e.g., the non-complementary strand of the target nucleic acid molecule. The RuvC domain is assembled from the three split RuvC motifs (RuvCI, RuvCII, and RuvCIII, which are often commonly referred to in the art as RuvCI domain, or N-terminal RuvC domain, RuvCII domain, and RuvCIII domain) at amino acids 1-59, 718-769, and 909-1098, respectively, of the sequence of S. pyogenes Cas9. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, however in the tertiary structure, the three RuvC motifs assemble and form the RuvC domain. The HNH domain shares structural similarity with HNH endonucleases, and cleaves a single strand, e.g., the complementary strand of the target nucleic acid molecule. The HNH domain lies between the RuvC II-III motifs and comprises amino acids 775-908 of the sequence of S. pyogenes Cas9. The PI domain interacts with the PAM of the target nucleic acid molecule, and comprises amino acids 1099-1368 of the sequence of S. pyogenes Cas9.
• A RuvC-Like Domain and an HNH-Like Domain
• In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises an HNH-like domain and a RuvC-like domain. In an embodiment, cleavage activity is dependent on a RuvC-like domain and an HNH-like domain. A Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can comprise one or more of the following domains: a RuvC-like domain and an HNH-like domain. In an embodiment, a Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide and the eaCas9 molecule or eaCas9 polypeptide comprises a RuvC-like domain, e.g., a RuvC-like domain described below, and/or an HNH-like domain, e.g., an HNH-like domain described below.
• RuvC-Like Domains
• In an embodiment, a RuvC-like domain cleaves, a single strand, e.g., the non-complementary strand of the target nucleic acid molecule. The Cas9 molecule or Cas9 polypeptide can include more than one RuvC-like domain (e.g., one, two, three or more RuvC-like domains). In an embodiment, a RuvC-like domain is at least 5, 6, 7, 8 amino acids in length but not more than 20, 19, 18, 17, 16 or 15 amino acids in length. In an embodiment, the Cas9 molecule or Cas9 polypeptide comprises an N-terminal RuvC-like domain of about 10 to 20 amino acids, e.g., about 15 amino acids in length.
• N-Terminal RuvC-Like Domains
• Some naturally occurring Cas9 molecules comprise more than one RuvC-like domain with cleavage being dependent on the N-terminal RuvC-like domain. Accordingly, Cas9 molecules or Cas9 polypeptide can comprise an N-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains are described below.
• In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula I:

• (SEQ ID NO: 8)

  D-X1-G-X2-X3-X4-X5-G-X6-X7-X8-X9,

• wherein,
• X1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);
• X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
• X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
• X4 is selected from S, Y, N and F (e.g., S);
• X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);
• X6 is selected from W, F, V, Y, S and L (e.g., W);
• X7 is selected from A, S, C, V and G (e.g., selected from A and S);
• X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and
• X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R, or, e.g., selected from T, V, I, L and Δ).
• In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO:8, by as many as 1 but no more than 2, 3, 4, or 5 residues.
• In embodiment, the N-terminal RuvC-like domain is cleavage competent.
• In embodiment, the N-terminal RuvC-like domain is cleavage incompetent.
• In an embodiment, a eaCas9 molecule or eaCas9 polypeptide comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula II:

• (SEQ ID NO: 9)

  D-X1-G-X2-X3-S-X5-G-X6-X7-X8-X9,,

• wherein
• X1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);
• X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
• X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
• X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);
• X6 is selected from W, F, V, Y, S and L (e.g., W);
• X7 is selected from A, S, C, V and G (e.g., selected from A and S);
• X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and
• X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R or selected from e.g., T, V, I, L and Δ).
• In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO:9 by as many as 1 but no more than 2, 3, 4, or 5 residues.
• In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:

• (SEQ ID NO: 10)

  D-I-G-X2-X3-S-V-G-W-A-X8-X9,

• wherein
• X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
• X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
• X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and
• X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R or selected from e.g., T, V, I, L and Δ).
• In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO:10 by as many as 1 but no more than, 2, 3, 4, or 5 residues.
• In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:

• (SEQ ID NO: 11)

  D-I-G-T-N-S-V-G-W-A-V-X,

• wherein
• X is a non-polar alkyl amino acid or a hydroxyl amino acid, e.g., X is selected from V, I, L and T (e.g., the eaCas9 molecule can comprise an N-terminal RuvC-like domain shown in FIGS. 2A-2G (is depicted as Y)).
• In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO:11 by as many as 1 but no more than, 2, 3, 4, or 5 residues.
• In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N-terminal RuvC like domain disclosed herein, e.g., in FIGS. 3A-3B or FIGS. 7A-7B, as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, 3 or all of the highly conserved residues identified in FIGS. 3A-3B or FIGS. 7A-7B are present.
• In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N-terminal RuvC-like domain disclosed herein, e.g., in FIGS. 4A-4B or FIGS. 7A-7B, as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, or all of the highly conserved residues identified in FIGS. 4A-4B or FIGS. 7A-7B are present.
• Additional RuvC-Like Domains
• In addition to the N-terminal RuvC-like domain, the Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can comprise one or more additional RuvC-like domains. In an embodiment, the Cas9 molecule or Cas9 polypeptide can comprise two additional RuvC-like domains. Preferably, the additional RuvC-like domain is at least 5 amino acids in length and, e.g., less than 15 amino acids in length, e.g., 5 to 10 amino acids in length, e.g., 8 amino acids in length.
• An additional RuvC-like domain can comprise an amino acid sequence:
• I-X1-X2-E-X3-A-R-E (SEQ ID NO:12), wherein
• X1 is V or H,
• X2 is I, L or V (e.g., I or V); and
• X3 is M or T.
• In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:
• I—V-X2-E-M-A-R-E (SEQ ID NO:13), wherein
• X2 is I, L or V (e.g., I or V) (e.g., the eaCas9 molecule or eaCas9 polypeptide can comprise an additional RuvC-like domain shown in FIG. 2A-2G or FIGS. 7A-7B (depicted as B)).
• An additional RuvC-like domain can comprise an amino acid sequence:
• H-H-A-X1-D-A-X2-X3 (SEQ ID NO: 14), wherein
• X1 is H or L;
• X2 is R or V; and
• X3 is E or V.
• In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:

• (SEQ ID NO: 15)

  H-H-A-H-D-A-Y-L.

• In an embodiment, the additional RuvC-like domain differs from a sequence of SEQ ID NO: 12, 13, 14 or 15 by as many as 1 but no more than 2, 3, 4, or 5 residues.
• In some embodiments, the sequence flanking the N-terminal RuvC-like domain is a sequence of formula V:

• (SEQ ID NO: 16)

  K-X1′-Y-X2′-X3′-X4′-Z-T-D-X9′-Y,.

  
  wherein
• X1′ is selected from K and P,
• X2′ is selected from V, L, I, and F (e.g., V, I and L);
• X3′ is selected from G, A and S (e.g., G),
• X4′ is selected from L, I, V and F (e.g., L);
• X9′ is selected from D, E, N and Q; and
• Z is an N-terminal RuvC-like domain, e.g., as described above.
• HNH-Like Domains
• In an embodiment, an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand of a double stranded nucleic acid molecule. In an embodiment, an HNH-like domain is at least 15, 20, 25 amino acids in length but not more than 40, 35 or 30 amino acids in length, e.g., 20 to 35 amino acids in length, e.g., 25 to 30 amino acids in length. Exemplary HNH-like domains are described below.
• In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain having an amino acid sequence of formula VI:
• X1-X2-X3-H-X4-X5-P-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-N-X16-X17-X18-X19-X20-X21-X22-X23-N(SEQ ID NO: 17), wherein
• X1 is selected from D, E, Q and N (e.g., D and E);
• X2 is selected from L, I, R, Q, V, M and K;
• X3 is selected from D and E;
• X4 is selected from I, V, T, A and L (e.g., A, I and V);
• X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);
• X6 is selected from Q, H, R, K, Y, I, L, F and W;
• X7 is selected from S, A, D, T and K (e.g., S and A);
• X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);
• X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;
• X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
• X11 is selected from D, S, N, R, L and T (e.g., D);
• X12 is selected from D, N and S;
• X13 is selected from S, A, T, G and R (e.g., S);
• X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
• X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
• X16 is selected from K, L, R, M, T and F (e.g., L, R and K);
• X17 is selected from V, L, I, A and T;
• X18 is selected from L, I, V and A (e.g., L and I);
• X19 is selected from T, V, C, E, S and A (e.g., T and V);
• X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
• X21 is selected from S, P, R, K, N, A, H, Q, G and L;
• X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and
• X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.
• In an embodiment, a HNH-like domain differs from a sequence of SEQ ID NO: 17 by at least one but no more than, 2, 3, 4, or 5 residues.
• In an embodiment, the HNH-like domain is cleavage competent.
• In an embodiment, the HNH-like domain is cleavage incompetent.
• In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain comprising an amino acid sequence of formula VII:

• (SEQ ID NO: 18)

  X1-X2-X3-H-X4-X5-P-X6-S-X8-X9-X10-D-D-S-X14-X15-N-

  K-V-L-X19-X20-X21-X22-X23-N,

• wherein
• X1 is selected from D and E;
• X2 is selected from L, I, R, Q, V, M and K;
• X3 is selected from D and E;
• X4 is selected from I, V, T, A and L (e.g., A, I and V);
• X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);
• X6 is selected from Q, H, R, K, Y, I, L, F and W;
• X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);
• X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;
• X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
• X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
• X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
• X19 is selected from T, V, C, E, S and A (e.g., T and V);
• X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
• X21 is selected from S, P, R, K, N, A, H, Q, G and L;
• X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and
• X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.
• In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 18 by 1, 2, 3, 4, or 5 residues.
• In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain comprising an amino acid sequence of formula VII:

• (SEQ ID NO: 19)

  X1-V-X3-H-I-V-P-X6-S-X8-X9-X10-D-D-S-X14-X15-N-K-

  V-L-T-X20-X21-X22-X23-N,

• wherein
• X1 is selected from D and E;
• X3 is selected from D and E;
• X6 is selected from Q, H, R, K, Y, I, L and W;
• X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);
• X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;
• X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
• X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
• X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
• X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
• X21 is selected from S, P, R, K, N, A, H, Q, G and L;
• X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and
• X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.
• In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 19 by 1, 2, 3, 4, or 5 residues.
• In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an HNH-like domain having an amino acid sequence of formula VIII:

• (SEQ ID NO: 20)

  D-X2-D-H-I-X5-P-Q-X7-F-X9-X10-D-X12-S-I-D-N-X16-V-

  L-X19-X20-S-X22-X23-N,

• wherein
• X2 is selected from I and V;
• X5 is selected from I and V;
• X7 is selected from A and S;
• X9 is selected from I and L;
• X10 is selected from K and T;
• X12 is selected from D and N;
• X16 is selected from R, K and L; X19 is selected from T and V;
• X20 is selected from S and R;
• X22 is selected from K, D and A; and
• X23 is selected from E, K, G and N (e.g., the eaCas9 molecule or eaCas9 polypeptide can comprise an HNH-like domain as described herein).
• In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 20 by as many as 1 but no more than 2, 3, 4, or 5 residues.
• In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises the amino acid sequence of formula IX:

• (SEQ ID NO: 21)

  L-Y-Y-L-Q-N-G-X1′-D-M-Y-X2′-X3′-X4′-X5′-L-D-I-X6′-

  X7′-L-S-X8′-Y-Z-N-R-X9′-K-X10′-D-X11′-V-P,

• wherein
• X1′ is selected from K and R;
• X2′ is selected from V and T;
• X3′ is selected from G and D;
• X4′ is selected from E, Q and D;
• X5′ is selected from E and D;
• X6′ is selected from D, N and H;
• X7′ is selected from Y, R and N;
• X8′ is selected from Q, D and N; X9′ is selected from G and E;
• X10′ is selected from S and G;
• X11′ is selected from D and N; and
• Z is an HNH-like domain, e.g., as described above.
• In an embodiment, the eaCas9 molecule or eaCas9 polypeptide comprises an amino acid sequence that differs from a sequence of SEQ ID NO:21 by as many as 1 but no more than 2, 3, 4, or 5 residues.
• In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in FIGS. 5A-5C or FIGS. 7A-7B, as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment, 1 or both of the highly conserved residues identified in FIGS. 5A-5C or FIGS. 7A-7B are present.
• In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in FIGS. 6A-6B or FIGS. 7A-7B, as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, all 3 of the highly conserved residues identified in FIGS. 6A-6B or FIGS. 7A-7B are present.
• Cas9 Activities
• Nuclease and Helicase Activities
• In an embodiment, the Cas9 molecule or Cas9 polypeptide is capable of cleaving a target nucleic acid molecule. Typically wild type Cas9 molecules cleave both strands of a target nucleic acid molecule. Cas9 molecules and Cas9 polypeptides can be engineered to alter nuclease cleavage (or other properties), e.g., to provide a Cas9 molecule or Cas9 polypeptide which is a nickase, or which lacks the ability to cleave target nucleic acid. A Cas9 molecule or Cas9 polypeptide that is capable of cleaving a target nucleic acid molecule is referred to herein as an eaCas9 (an enzymatically active Cas9) molecule or eaCas9 polypeptide. In an embodiment, an eaCas9 molecule or Cas9 polypeptide comprises one or more of the following activities:
• a nickase activity, i.e., the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule;
• a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;
• an endonuclease activity;
• an exonuclease activity; and
• a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid.
• In an embodiment, an enzymatically active Cas9 or an eaCas9 molecule or an eaCas9 polypeptide cleaves both DNA strands and results in a double stranded break. In an embodiment, an eaCas9 molecule cleaves only one strand, e.g., the strand to which the gRNA hybridizes to, or the strand complementary to the strand the gRNA hybridizes with. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an HNH-like domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with an HNH-like domain and cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an active, or cleavage competent, HNH-like domain and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an inactive, or cleavage incompetent, HNH-like domain and an active, or cleavage competent, N-terminal RuvC-like domain.
• Some Cas9 molecules or Cas9 polypeptides have the ability to interact with a gRNA molecule, and in conjunction with the gRNA molecule localize to a core target domain, but are incapable of cleaving the target nucleic acid, or incapable of cleaving at efficient rates. Cas9 molecules having no, or no substantial, cleavage activity are referred to herein as an eiCas9 molecule or eiCas9 polypeptide. For example, an eiCas9 molecule or eiCas9 polypeptide can lack cleavage activity or have substantially less, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule or eiCas9 polypeptide, as measured by an assay described herein.
• Targeting and PAMs
• A Cas9 molecule or Cas9 polypeptide, is a polypeptide that can interact with a guide RNA (gRNA) molecule and, in concert with the gRNA molecule, localizes to a site which comprises a target domain and PAM sequence.
• In an embodiment, the ability of an eaCas9 molecule or eaCas9 polypeptide to interact with and cleave a target nucleic acid is PAM sequence dependent. A PAM sequence is a sequence in the target nucleic acid. In an embodiment, cleavage of the target nucleic acid occurs upstream from the PAM sequence. EaCas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In an embodiment, an eaCas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Mali et al., SCIENCE 2013; 339(6121): 823-826. In an embodiment, an eaCas9 molecule of S. thermophilus recognizes the sequence motif NGGNG and NNAGAAW (W=A or T) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from these sequences. See, e.g., Horvath et al., SCIENCE 2010; 327(5962):167-170, and Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. mutans recognizes the sequence motif NGG and/or NAAR (R=A or G) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence. See, e.g., Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRR (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRN (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRT (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRRV (R=A or G, V=A, G or C) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of Neisseria meningitidis recognizes the sequence motif NNNNGATT or NNNGCTT and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Hou et al., PNAS Early Edition 2013, 1-6. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using a transformation assay described in Jinek et al., SCIENCE 2012 337:816. In the aforementioned embodiments, N can be any nucleotide residue, e.g., any of A, G, C or T.
• As is discussed herein, Cas9 molecules can be engineered to alter the PAM specificity of the Cas9 molecule.
• Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA BIOLOGY 2013 10:5, 727-737. Such Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 10 bacterial family, a cluster 11 bacterial family, a cluster 12 bacterial family, a cluster 13 bacterial family, a cluster 14 bacterial family, a cluster 15 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26 bacterial family, a cluster 27 bacterial family, a cluster 28 bacterial family, a cluster 29 bacterial family, a cluster 30 bacterial family, a cluster 31 bacterial family, a cluster 32 bacterial family, a cluster 33 bacterial family, a cluster 34 bacterial family, a cluster 35 bacterial family, a cluster 36 bacterial family, a cluster 37 bacterial family, a cluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40 bacterial family, a cluster 41 bacterial family, a cluster 42 bacterial family, a cluster 43 bacterial family, a cluster 44 bacterial family, a cluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47 bacterial family, a cluster 48 bacterial family, a cluster 49 bacterial family, a cluster 50 bacterial family, a cluster 51 bacterial family, a cluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54 bacterial family, a cluster 55 bacterial family, a cluster 56 bacterial family, a cluster 57 bacterial family, a cluster 58 bacterial family, a cluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61 bacterial family, a cluster 62 bacterial family, a cluster 63 bacterial family, a cluster 64 bacterial family, a cluster 65 bacterial family, a cluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68 bacterial family, a cluster 69 bacterial family, a cluster 70 bacterial family, a cluster 71 bacterial family, a cluster 72 bacterial family, a cluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75 bacterial family, a cluster 76 bacterial family, a cluster 77 bacterial family, or a cluster 78 bacterial family.
• Exemplary naturally occurring Cas9 molecules include a Cas9 molecule of a cluster 1 bacterial family. Examples include a Cas9 molecule of: S. pyogenes (e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans (e.g., strain UA159, NN2025), S. macacae (e.g., strain NCTC11558), S. gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. anginosus (e.g., strain F0211), S. agalactiae (e.g., strain NEM316, A909), Listeria monocytogenes (e.g., strain F6854), Listeria innocua (L. innocua, e.g., strain Clip11262), Enterococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium (e.g., strain 1,231,408). Additional exemplary Cas9 molecules are a Cas9 molecule of Neisseria meningitides (Hou et al., PNAS Early Edition 2013, 1-6 and a S. aureus cas9 molecule.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence:
• having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with;
• differs at no more than, 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues when compared with;
• differs by at least 1, 2, 5, 10 or 20 amino acids, but by no more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or
• is identical to any Cas9 molecule sequence described herein, or a naturally occurring Cas9 molecule sequence, e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al., RNA BIOLOGY 2013 10:5, 727-737; Hou et al., PNAS Early Edition 2013, 1-6; SEQ ID NO:1-4. In an embodiment, the Cas9 molecule or Cas9 polypeptide comprises one or more of the following activities: a nickase activity; a double stranded cleavage activity (e.g., an endonuclease and/or exonuclease activity); a helicase activity; or the ability, together with a gRNA molecule, to localize to a target nucleic acid.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises any of the amino acid sequence of the consensus sequence of FIGS. 2A-2G, wherein “*” indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, S. thermophilus, S. mutans and L. innocua, and “-” indicates any amino acid. In an embodiment, a Cas9 molecule or Cas9 polypeptide differs from the sequence of the consensus sequence disclosed in FIGS. 2A-2G by at least 1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises the amino acid sequence of SEQ ID NO:7 of FIGS. 7A-7B, wherein “*” indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, or N. meningitides, “-” indicates any amino acid, and “-” indicates any amino acid or absent. In an embodiment, a Cas9 molecule or Cas9 polypeptide differs from the sequence of SEQ ID NO:6 or 7 disclosed in FIGS. 7A-7B by at least 1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.
• A comparison of the sequence of a number of Cas9 molecules indicate that certain regions are conserved. These are identified below as:
• region 1 (residues 1 to 180, or in the case of region 1′ residues 120 to 180)
• region 2 (residues 360 to 480);
• region 3 (residues 660 to 720);
• region 4 (residues 817 to 900); and
• region 5 (residues 900 to 960);
• In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises regions 1-5, together with sufficient additional Cas9 molecule sequence to provide a biologically active molecule, e.g., a Cas9 molecule having at least one activity described herein. In an embodiment, each of regions 1-5, independently, have 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the corresponding residues of a Cas9 molecule or Cas9 polypeptide described herein, e.g., a sequence from FIGS. 2A-2G or from FIGS. 7A-7B.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 1:
• having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 1-180 (the numbering is according to the motif sequence in FIG. 2; 52% of residues in the four Cas9 sequences in FIGS. 2A-2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes;
• differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than 90, 80, 70, 60, 50, 40 or 30 amino acids from amino acids 1-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or Listeria innocua; or
• is identical to 1-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 1′:
• having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 120-180 (55% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;
• differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 120-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua; or
• is identical to 120-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 2:
• having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 360-480 (52% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;
• differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua; or
• is identical to 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 3:
• having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with amino acids 660-720 (56% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;
• differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua; or
• is identical to 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 4:
• having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with amino acids 817-900 (55% of residues in the four Cas9 sequences in FIGS. 2A-2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;
• differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 817-900 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua; or
• is identical to 817-900 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, comprises an amino acid sequence referred to as region 5:
• having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with amino acids 900-960 (60% of residues in the four Cas9 sequences in FIGS. 2A-2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;
• differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 900-960 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua; or
• is identical to 900-960 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua.
• Engineered or Altered Cas9 Molecules and Cas9 Polypeptides
• Cas9 molecules and Cas9 polypeptides described herein, e.g., naturally occurring Cas9 molecules, can possess any of a number of properties, including: nickase activity, nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to associate functionally with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity). In an embodiment, a Cas9 molecule or Cas9 polypeptide can include all or a subset of these properties. In typical embodiments, a Cas9 molecule or Cas9 polypeptide have the ability to interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site in a nucleic acid. Other activities, e.g., PAM specificity, cleavage activity, or helicase activity can vary more widely in Cas9 molecules and Cas9 polypeptides.
• Cas9 molecules include engineered Cas9 molecules and engineered Cas9 polypeptides (engineered, as used in this context, means merely that the Cas9 molecule or Cas9 polypeptide differs from a reference sequences, and implies no process or origin limitation). An engineered Cas9 molecule or Cas9 polypeptide can comprise altered enzymatic properties, e.g., altered nuclease activity, (as compared with a naturally occurring or other reference Cas9 molecule) or altered helicase activity. As discussed herein, an engineered Cas9 molecule or Cas9 polypeptide can have nickase activity (as opposed to double strand nuclease activity). In an embodiment an engineered Cas9 molecule or Cas9 polypeptide can have an alteration that alters its size, e.g., a deletion of amino acid sequence that reduces its size, e.g., without significant effect on one or more, or any Cas9 activity. In an embodiment, an engineered Cas9 molecule or Cas9 polypeptide can comprise an alteration that affects PAM recognition. E.g., an engineered Cas9 molecule can be altered to recognize a PAM sequence other than that recognized by the endogenous wild-type PI domain. In an embodiment, a Cas9 molecule or Cas9 polypeptide can differ in sequence from a naturally occurring Cas9 molecule but not have significant alteration in one or more Cas9 activities.
• Cas9 molecules or Cas9 polypeptides with desired properties can be made in a number of ways, e.g., by alteration of a parental, e.g., naturally occurring Cas9 molecules or Cas9 polypeptides to provide an altered Cas9 molecule or Cas9 polypeptide having a desired property. For example, one or more mutations or differences relative to a parental Cas9 molecule, e.g., a naturally occurring or engineered Cas9 molecule, can be introduced. Such mutations and differences comprise: substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids); insertions; or deletions. In an embodiment, a Cas9 molecule or Cas9 polypeptide can comprises one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations, but less than 200, 100, or 80 mutations relative to a reference, e.g., a parental, Cas9 molecule.
• In an embodiment, a mutation or mutations do not have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein. In an embodiment, a mutation or mutations have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein.
• Non-Cleaving and Modified-Cleavage Cas9 Molecules and Cas9 Polypeptides
• In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule or Cas9 polypeptide can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S. pyogenes, as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded nucleic acid (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.
• Modified Cleavage eaCas9 Molecules and eaCas9 Polypeptides
• In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises one or more of the following activities: cleavage activity associated with an N-terminal RuvC-like domain; cleavage activity associated with an HNH-like domain; cleavage activity associated with an HNH-like domain and cleavage activity associated with an N-terminal RuvC-like domain.
• In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an active, or cleavage competent, HNH-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21) and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. An exemplary inactive, or cleavage incompetent N-terminal RuvC-like domain can have a mutation of an aspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in FIGS. 2A-2G or an aspartic acid at position 10 of SEQ ID NO: 7, e.g., can be substituted with an alanine. In an embodiment, the eaCas9 molecule or eaCas9 polypeptide differs from wild type in the N-terminal RuvC-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.
• In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises an inactive, or cleavage incompetent, HNH domain and an active, or cleavage competent, N-terminal RuvC-like domain (e.g., a RuvC-like domain described herein, e.g., SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16). Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a histidine shown at position 856 of the consensus sequence disclosed in FIGS. 2A-2G, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine shown at position 870 of the consensus sequence disclosed in FIGS. 2A-2G and/or at position 879 of the consensus sequence disclosed in FIGS. 2A-2G, e.g., can be substituted with an alanine. In an embodiment, the eaCas9 differs from wild type in the HNH-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.
• Alterations in the Ability to Cleave One or Both Strands of a Target Nucleic Acid
• In an embodiment, exemplary Cas9 activities comprise one or more of PAM specificity, cleavage activity, and helicase activity. A mutation(s) can be present, e.g., in one or more RuvC-like domain, e.g., an N-terminal RuvC-like domain; an HNH-like domain; a region outside the RuvC-like domains and the HNH-like domain. In some embodiments, a mutation(s) is present in a RuvC-like domain, e.g., an N-terminal RuvC-like domain. In some embodiments, a mutation(s) is present in an HNH-like domain. In some embodiments, mutations are present in both a RuvC-like domain, e.g., an N-terminal RuvC-like domain and an HNH-like domain.
• Exemplary mutations that may be made in the RuvC domain or HNH domain with reference to the S. pyogenes sequence include: D10A, E762A, H840A, N854A, N863A and/or D986A.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide is an eiCas9 molecule or eiCas9 polypeptide comprising one or more differences in a RuvC domain and/or in an HNH domain as compared to a reference Cas9 molecule, and the eiCas9 molecule or eiCas9 polypeptide does not cleave a nucleic acid, or cleaves with significantly less efficiency than does wildtype, e.g., when compared with wild type in a cleavage assay, e.g., as described herein, cuts with less than 50, 25, 10, or 1% of a reference Cas9 molecule, as measured by an assay described herein.
• Whether or not a particular sequence, e.g., a substitution, may affect one or more activity, such as targeting activity, cleavage activity, etc., can be evaluated or predicted, e.g., by evaluating whether the mutation is conservative or by the method described in Section IV. In an embodiment, a “non-essential” amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be altered from the wild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (e.g., cleavage activity), whereas changing an “essential” amino acid residue results in a substantial loss of activity (e.g., cleavage activity).
• In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule or Cas9 polypeptide can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S aureus, S. pyogenes, or C. jejuni as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded break (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S aureus, S. pyogenes, or C. jejuni); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complimentary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S aureus, S. pyogenes, or C. jejuni); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising one or more of the following activities: cleavage activity associated with a RuvC domain; cleavage activity associated with an HNH domain; cleavage activity associated with an HNH domain and cleavage activity associated with a RuvC domain.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eiCas9 molecule or eiCas9 polypeptide which does not cleave a nucleic acid molecule (either double stranded or single stranded nucleic acid molecules) or cleaves a nucleic acid molecule with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can be a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni or N. meningitidis. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology. In an embodiment, the eiCas9 molecule or eiCas9 polypeptide lacks substantial cleavage activity associated with a RuvC domain and cleavage activity associated with an HNH domain.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in FIGS. 2A-2G, and has one or more amino acids that differ from the amino acid sequence of S. pyogenes (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an “-” in the consensus sequence disclosed in FIGS. 2A-2G or SEQ ID NO: 7.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide comprises a sequence in which:
• the sequence corresponding to the fixed sequence of the consensus sequence disclosed in FIGS. 2A-2G differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in FIGS. 2A-2G;
• • the sequence corresponding to the residues identified by “*” in the consensus sequence disclosed in FIGS. 2A-2G differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule; and, the sequence corresponding to the residues identified by “-” in the consensus sequence disclosed in FIGS. 2A-2G differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “-” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in FIGS. 2A-2G, and has one or more amino acids that differ from the amino acid sequence of S. thermophilus (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an “-” in the consensus sequence disclosed in FIGS. 2A-2G.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide comprises a sequence in which:
• the sequence corresponding to the fixed sequence of the consensus sequence disclosed in FIGS. 2A-2G differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in FIGS. 2A-2G;
• the sequence corresponding to the residues identified by “*” in the consensus sequence disclosed in FIGS. 2A-2G differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule; and,
• the sequence corresponding to the residues identified by “-” in the consensus sequence disclosed in FIGS. 2A-2G differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “-” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in FIGS. 2A-2G, and has one or more amino acids that differ from the amino acid sequence of S. mutans (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an “-” in the consensus sequence disclosed in FIGS. 2A-2G.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide comprises a sequence in which:
• the sequence corresponding to the fixed sequence of the consensus sequence disclosed in FIGS. 2A-2G differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in FIGS. 2A-2G;
• the sequence corresponding to the residues identified by “*” in the consensus sequence disclosed in FIGS. 2A-2G differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutans Cas9 molecule; and,
• the sequence corresponding to the residues identified by “-” in the consensus sequence disclosed in FIGS. 2A-2G differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “-” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutans Cas9 molecule.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acid residues of L. innocula shown in the consensus sequence disclosed in FIGS. 2A-2G, and has one or more amino acids that differ from the amino acid sequence of L. innocula (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an “-” in the consensus sequence disclosed in FIGS. 2A-2G.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide comprises a sequence in which:
• the sequence corresponding to the fixed sequence of the consensus sequence disclosed in FIGS. 2A-2G differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in FIGS. 2A-2G;
• the sequence corresponding to the residues identified by “*” in the consensus sequence disclosed in FIGS. 2A-2G differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule; and,
• the sequence corresponding to the residues identified by “-” in the consensus sequence disclosed in FIGS. 2A-2G differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “-” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule.
• In an embodiment, the altered Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, can be a fusion, e.g., of two of more different Cas9 molecules, e.g., of two or more naturally occurring Cas9 molecules of different species. For example, a fragment of a naturally occurring Cas9 molecule of one species can be fused to a fragment of a Cas9 molecule of a second species. As an example, a fragment of a Cas9 molecule of S. pyogenes comprising an N-terminal RuvC-like domain can be fused to a fragment of Cas9 molecule of a species other than S. pyogenes (e.g., S. thermophilus) comprising an HNH-like domain.
• Cas9 Molecules and Cas9 Polypeptides with Altered PAM Recognition or No PAM Recognition
• Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example, the PAM recognition sequences described above for S. pyogenes, S. thermophiles, S. mutans, S. aureus and N. meningitides.
• In an embodiment, a Cas9 molecule or Cas9 polypeptide has the same PAM specificities as a naturally occurring Cas9 molecule. In other embodiments, a Cas9 molecule or Cas9 polypeptide has a PAM specificity not associated with a naturally occurring Cas9 molecule, or a PAM specificity not associated with the naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, a naturally occurring Cas9 molecule can be altered, e.g., to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9 molecule recognizes to decrease off target sites and/or improve specificity; or eliminate a PAM recognition requirement. In an embodiment, a Cas9 molecule or Cas9 polypeptide can be altered, e.g., to increase length of PAM recognition sequence and/or improve Cas9 specificity to high level of identity (e.g., 98%, 99% or 100% match between gRNA and a PAM sequence), e.g., to decrease off target sites and increase specificity. In an embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. In an embodiment, the Cas9 specificity requires at least 90%, 95%, 96%, 97%, 98%, 99% or more homology between the gRNA and the PAM sequence. Cas9 molecules or Cas9 polypeptides that recognize different PAM sequences and/or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas9 molecules are described, e.g., in Esvelt et al. NATURE 2011, 472(7344): 499-503. Candidate Cas9 molecules can be evaluated, e.g., by methods described in Section IV.
• Alterations of the PI domain, which mediates PAM recognition, are discussed below.
• Synthetic Cas9 Molecules and Cas9 Polypeptides with Altered PI Domains
• Current genome-editing methods are limited in the diversity of target sequences that can be targeted by the PAM sequence that is recognized by the Cas9 molecule utilized. A synthetic Cas9 molecule (or Syn-Cas9 molecule), or synthetic Cas9 polypeptide (or Syn-Cas9 polypeptide), as that term is used herein, refers to a Cas9 molecule or Cas9 polypeptide that comprises a Cas9 core domain from one bacterial species and a functional altered PI domain, i.e., a PI domain other than that naturally associated with the Cas9 core domain, e.g., from a different bacterial species.
• In an embodiment, the altered PI domain recognizes a PAM sequence that is different from the PAM sequence recognized by the naturally-occurring Cas9 from which the Cas9 core domain is derived. In an embodiment, the altered PI domain recognizes the same PAM sequence recognized by the naturally-occurring Cas9 from which the Cas9 core domain is derived, but with different affinity or specificity. A Syn-Cas9 molecule or Syn-Cas9 polypeptide can be, respectively, a Syn-eaCas9 molecule or Syn-eaCas9 polypeptide or a Syn-eiCas9 molecule Syn-eiCas9 polypeptide.
• An exemplary Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises:
• a) a Cas9 core domain, e.g., a Cas9 core domain from Table 8 or 9, e.g., a S. aureus, S. pyogenes, or C. jejuni Cas9 core domain; and
• b) an altered PI domain from a species X Cas9 sequence selected from Tables 11 and 12.
• In an embodiment, the RKR motif (the PAM binding motif) of said altered PI domain comprises: differences at 1, 2, or 3 amino acid residues; a difference in amino acid sequence at the first, second, or third position; differences in amino acid sequence at the first and second positions, the first and third positions, or the second and third positions; as compared with the sequence of the RKR motif of the native or endogenous PI domain associated with the Cas9 core domain.
• In an embodiment, the Cas9 core domain comprises the Cas9 core domain from a species X Cas9 from Table 8 and said altered PI domain comprises a PI domain from a species Y Cas9 from Table 8.
• In an embodiment, the RKR motif of the species X Cas9 is other than the RKR motif of the species Y Cas9.
• In an embodiment, the RKR motif of the altered PI domain is selected from XXY, XNG, and XNQ.
• In an embodiment, the altered PI domain has at least 60, 70, 80, 90, 95, or 100% homology with the amino acid sequence of a naturally occurring PI domain of said species Y from Table 8.
• In an embodiment, the altered PI domain differs by no more than 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from the amino acid sequence of a naturally occurring PI domain of said second species from Table 8.
• In an embodiment, the Cas9 core domain comprises a S. aureus core domain and altered PI domain comprises: an A. denitrificans PI domain; a C. jejuni PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table 12.
• In an embodiment, the Cas9 core domain comprises a S. pyogenes core domain and the altered PI domain comprises: an A. denitrificans PI domain; a C. jejuni PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table 12.
• In an embodiment, the Cas9 core domain comprises a C. jejuni core domain and the altered PI domain comprises: an A. denitrificans PI domain; a H. mustelae PI domain; or an altered PI domain of species X PI domain, wherein species X is selected from Table 12.
• In an embodiment, the Cas9 molecule or Cas9 polypeptide further comprises a linker disposed between said Cas9 core domain and said altered PI domain.
• In an embodiment, the linker comprises: a linker described elsewhere herein disposed between the Cas9 core domain and the heterologous PI domain. Suitable linkers are further described in Section V.
• Exemplary altered PI domains for use in Syn-Cas9 molecules are described in Tables 11 and 12. The sequences for the 83 Cas9 orthologs referenced in Tables 11 and 12 are provided in Table 8. Table 10 provides the Cas9 orthologs with known PAM sequences and the corresponding RKR motif.
• In an embodiment, a Syn-Cas9 molecule or Syn-Cas9 polypeptide may also be size-optimized, e.g., the Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises one or more deletions, and optionally one or more linkers disposed between the amino acid residues flanking the deletions. In an embodiment, a Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises a REC deletion.
• Size-Optimized Cas9 Molecules and Cas9 Polypeptides
• Engineered Cas9 molecules and engineered Cas9 polypeptides described herein include a Cas9 molecule or Cas9 polypeptide comprising a deletion that reduces the size of the molecule while still retaining desired Cas9 properties, e.g., essentially native conformation, Cas9 nuclease activity, and/or target nucleic acid molecule recognition. Provided herein are Cas9 molecules or Cas9 polypeptides comprising one or more deletions and optionally one or more linkers, wherein a linker is disposed between the amino acid residues that flank the deletion. Methods for identifying suitable deletions in a reference Cas9 molecule, methods for generating Cas9 molecules with a deletion and a linker, and methods for using such Cas9 molecules will be apparent to one of ordinary skill in the art upon review of this document.
• A Cas9 molecule, e.g., a S. aureus, S. pyogenes, or C. jejuni, Cas9 molecule, having a deletion is smaller, e.g., has reduced number of amino acids, than the corresponding naturally-occurring Cas9 molecule. The smaller size of the Cas9 molecules allows increased flexibility for delivery methods, and thereby increases utility for genome-editing. A Cas9 molecule or Cas9 polypeptide can comprise one or more deletions that do not substantially affect or decrease the activity of the resultant Cas9 molecules or Cas9 polypeptides described herein. Activities that are retained in the Cas9 molecules or Cas9 polypeptides comprising a deletion as described herein include one or more of the following:
• a nickase activity, i.e., the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule; a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;
• an endonuclease activity;
• an exonuclease activity;
• a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid;
• and recognition activity of a nucleic acid molecule, e.g., a target nucleic acid or a gRNA.
• Activity of the Cas9 molecules or Cas9 polypeptides described herein can be assessed using the activity assays described herein or in the art.
• Identifying Regions Suitable for Deletion
• Suitable regions of Cas9 molecules for deletion can be identified by a variety of methods. Naturally-occurring orthologous Cas9 molecules from various bacterial species, e.g., any one of those listed in Table 8, can be modeled onto the crystal structure of S. pyogenes Cas9 (Nishimasu et al., Cell, 156:935-949, 2014) to examine the level of conservation across the selected Cas9 orthologs with respect to the three-dimensional conformation of the protein. Less conserved or unconserved regions that are spatially located distant from regions involved in Cas9 activity, e.g., interface with the target nucleic acid molecule and/or gRNA, represent regions or domains are candidates for deletion without substantially affecting or decreasing Cas9 activity.
• REC-Optimized Cas9 Molecules and Cas9 Polypeptides
• A REC-optimized Cas9 molecule, or a REC-optimized Cas9 polypeptide, as that term is used herein, refers to a Cas9 molecule or Cas9 polypeptide that comprises a deletion in one or both of the REC2 domain and the RE1_CT domain (collectively a REC deletion), wherein the deletion comprises at least 10% of the amino acid residues in the cognate domain. A REC-optimized Cas9 molecule or Cas9 polypeptide can be an eaCas9 molecule or eaCas9 polypeptide, or an eiCas9 molecule or eiCas9 polypeptide. An exemplary REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises:
• a) a deletion selected from:
• • i) a REC2 deletion;
  • ii) a REC1_CT deletion; or
  • iii) a REC1_SUB deletion.
• Optionally, a linker is disposed between the amino acid residues that flank the deletion. In an embodiment, a Cas9 molecule or Cas9 polypeptide includes only one deletion, or only two deletions. A Cas9 molecule or Cas9 polypeptide can comprise a REC2 deletion and a REC1_CT deletion. A Cas9 molecule or Cas9 polypeptide can comprise a REC2 deletion and a REC1_SUB deletion.
• Generally, the deletion will contain at least 10% of the amino acids in the cognate domain, e.g., a REC2 deletion will include at least 10% of the amino acids in the REC2 domain.
• A deletion can comprise: at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the amino acid residues of its cognate domain; all of the amino acid residues of its cognate domain; an amino acid residue outside its cognate domain; a plurality of amino acid residues outside its cognate domain; the amino acid residue immediately N terminal to its cognate domain; the amino acid residue immediately C terminal to its cognate domain; the amino acid residue immediately N terminal to its cognate and the amino acid residue immediately C terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues N terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues C terminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15, or 20, amino acid residues N terminal to its cognate domain and a plurality of e.g., up to 5, 10, 15, or 20, amino acid residues C terminal to its cognate domain.
• In an embodiment, a deletion does not extend beyond: its cognate domain; the N terminal amino acid residue of its cognate domain; the C terminal amino acid residue of its cognate domain.
• A REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide can include a linker disposed between the amino acid residues that flank the deletion. Any linkers known in the art that maintain the conformation or native fold of the Cas9 molecule (thereby retaining Cas9 activity) can be used between the amino acid resides that flank a REC deletion in a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide. Linkers for use in generating recombinant proteins, e.g., multi-domain proteins, are known in the art (Chen et al., Adv Drug Delivery Rev, 65:1357-69, 2013).
• In an embodiment, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, other than any REC deletion and associated linker, has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% homology with the amino acid sequence of a naturally occurring Cas9, e.g., a Cas9 molecule described in Table 8, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.
• In an embodiment, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, other than any REC deletion and associated linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25, amino acid residues from the amino acid sequence of a naturally occurring Cas 9, e.g., a Cas9 molecule described in Table 8, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.
• In an embodiment, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, other than any REC deletion and associate linker, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25% of the, amino acid residues from the amino acid sequence of a naturally occurring Cas 9, e.g., a Cas9 molecule described in Table 8, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C. jejuni Cas9 molecule.
• For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).
• Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
• The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
• Sequence information for exemplary REC deletions are provided for 83 naturally-occurring Cas9 orthologs in Table 8.
• The amino acid sequences of exemplary Cas9 molecules from different bacterial species are shown below.

• TABLE 8
  Amino Acid Sequence of Cas9 Orthologs

  REC2

  REC1CT

  Recsub

  	Amino	start	stop	# AA	start	stop	# AA	start	stop	# AA
  	acid	(AA	(AA	deleted	(AA	(AA	deleted	(AA	(AA	deleted
  Species/Composite ID	sequence	pos)	pos)	(n)	pos)	pos)	(n)	pos)	pos)	(n)

  Staphylococcus Aureus	SEQ ID		126	166	41	296	352	57	296	352	57
  tr|J7RUA5|J7RUA5_STAAU	NO: 304
  Streptococcus Pyogenes	SEQ ID		176	314	139	511	592	82	511	592	82
  sp|Q99ZW2|CAS9_STRP1	NO: 305
  Campylobacter jejuni NCTC 11168	SEQ ID	137	181	45	316	360	45	316	360	45
  gi|218563121|ref|YP_002344900.1	NO: 306
  Bacteroides fragilis NCTC 9343	SEQ ID	148	339	192	524	617	84	524	617	84
  gi|60683389|ref|YP_213533.1|	NO: 307
  Bifidobacterium bifidum S17	SEQ ID	173	335	163	516	607	87	516	607	87
  gi|310286728|ref|YP_003937986.	NO: 308
  Veillonella atypica ACS-134-V-Col7a	SEQ ID	185	339	155	574	663	79	574	663	79
  gi|303229466|ref|ZP_07316256.1	NO: 309
  Lactobacillus rhamnosus GG	SEQ ID	169	320	152	559	645	78	559	645	78
  gi|258509199|ref|YP_003171950.1	NO: 310
  Filifactor alocis ATCC 35896	SEQ ID	166	314	149	508	592	76	508	592	76
  gi|374307738|ref|YP_005054169.1	NO: 311
  Oenococcus kitaharae DSM 17330	SEQ ID	169	317	149	555	639	80	555	639	80
  gi|366983953|gb|EHN59352.1|	NO: 312
  Fructobacillus fructosus KCTC 3544	SEQ ID	168	314	147	488	571	76	488	571	76
  gi|339625081|ref|ZP_08660870.1	NO: 313
  Catenibacterium mitsuokai DSM 15897	SEQ ID	173	318	146	511	594	78	511	594	78
  gi|224543312|ref|ZP_03683851.1	NO: 314
  Finegoldia magna ATCC 29328	SEQ ID	168	313	146	452	534	77	452	534	77
  gi|169823755|ref|YP_001691366.1	NO: 315
  CoriobacteriumglomeransPW2	SEQ ID	175	318	144	511	592	82	511	592	82
  gi|328956315|ref|YP_004373648.1	NO: 316
  Eubacterium yurii ATCC 43715	SEQ ID	169	310	142	552	633	76	552	633	76
  gi|306821691|ref|ZP_07455288.1	NO: 317
  Peptoniphilus duerdenii ATCC BAA-1640	SEQ ID	171	311	141	535	615	76	535	615	76
  gi|304438954|ref|ZP_07398877.1	NO: 318
  Acidaminococcus sp. D21	SEQ ID	167	306	140	511	591	75	511	591	75
  gi|227824983|ref|ZP_03989815.1	NO: 319
  Lactobacillus farciminis KCTC 3681	SEQ ID	171	310	140	542	621	85	542	621	85
  gi|336394882|ref|ZP_08576281.1	NO: 320
  Streptococcus sanguinis SK49	SEQ ID	185	324	140	411	490	85	411	490	85
  gi|422884106|ref|ZP_16930555.1	NO: 321
  Coprococcus catus GD-7	SEQ ID	172	310	139	556	634	76	556	634	76
  gi|291520705|emb|CBK78998.1|	NO: 322
  Streptococcus mutans UA159	SEQ ID	176	314	139	392	470	84	392	470	84
  gi|24379809|ref|NP_721764.1|	NO: 323
  Streptococcus pyogenes M1 GAS	SEQ ID	176	314	139	523	600	82	523	600	82
  gi|13622193|gb|AAK33936.1|	NO: 324
  Streptococcus thermophilus LMD-9	SEQ ID	176	314	139	481	558	81	481	558	81
  gi|116628213|ref|YP_820832.1|	NO: 325
  Fusobacteriumnucleatum ATCC49256	SEQ ID	171	308	138	537	614	76	537	614	76
  gi|34762592|ref|ZP_00143587.1|	NO: 326
  Planococcus antarcticus DSM 14505	SEQ ID	162	299	138	538	614	94	538	614	94
  gi|389815359|ref|ZP_10206685.1	NO: 327
  Treponema denticola ATCC 35405	SEQ ID	169	305	137	524	600	81	524	600	81
  gi|42525843|ref|NP_970941.1|	NO: 328
  Solobacterium moorei F0204	SEQ ID	179	314	136	544	619	77	544	619	77
  gi|320528778|ref|ZP_08029929.1	NO: 329
  Staphylococcus pseudintermedius ED99	SEQ ID	164	299	136	531	606	92	531	606	92
  gi|323463801|gb|ADX75954.1|	NO: 330
  Flavobacterium branchiophilum FL-15	SEQ ID	162	286	125	538	613	63	538	613	63
  gi|347536497|ref|YP_004843922.1	NO: 331
  Ignavibacterium album JCM 16511	SEQ ID	223	329	107	357	432	90	357	432	90
  gi|385811609|ref|YP_005848005.1	NO: 332
  Bergeyella zoohelcum ATCC 43767	SEQ ID	165	261	97	529	604	56	529	604	56
  gi|423317190|ref|ZP_17295095.1	NO: 333
  Nitrobacter hamburgensis X14	SEQ ID	169	253	85	536	611	48	536	611	48
  gi|92109262|ref|YP_571550.1|	NO: 334
  Odoribacter laneus YIT 12061	SEQ ID	164	242	79	535	610	63	535	610	63
  gi|374384763|ref|ZP_09642280.1	NO: 335
  Legionella pneumophila str. Paris	SEQ ID		164	239	76	402	476	67	402	476	67
  gi|54296138|ref|YP_122507.1|	NO: 336
  Bacteroides sp. 20 3	SEQ ID	198	269	72	530	604	83	530	604	83
  gi|301311869|ref|ZP_07217791.1	NO: 337
  Akkermansia muciniphila ATCC BAA-835	SEQ ID	136	202	67	348	418	62	348	418	62
  gi|187736489|ref|YP_001878601	NO: 338
  Prevotella sp. C561	SEQ ID		184	250	67	357	425	78	357	425	78
  gi|345885718|ref|ZP_08837074.1	NO: 339
  Wolinella succinogenes DSM 1740	SEQ ID	157	218	36	401	468	60	401	468	60
  gi|34557932|ref|NP_907747.1|	NO: 340
  Alicyclobacillus hesperidum URH17-3-68	SEQ ID	142	196	55	416	482	61	416	482	61
  gi|403744858|ref|ZP_10953934.1	NO: 341
  Caenispirillum salinarum AK4	SEQ ID		161	214	54	330	393	68	330	393	68
  gi|427429481|ref|ZP_18919511.1	NO: 342
  Eubacterium rectale ATCC 33656	SEQ ID	133	185	53	322	384	60	322	384	60
  gi|238924075|ref|YP_002937591.1	NO: 343
  Mycoplasma synoviae 53	SEQ ID	187	239	53	319	381	80	319	381	80
  gi|71894592|ref|YP_278700.1|	NO: 344
  Porphyromonas sp. oral taxon 279 str. F0450	SEQ ID	150	202	53	309	371	60	309	371	60
  gi|402847315|ref|ZP_10895610.1	NO: 345
  Streptococcus thermophilus LMD-9	SEQ ID	127	178	139	424	486	81	424	486	81
  gi|116627542|ref|YP_820161.1|	NO: 346
  Roseburia inulinivorans DSM 16841	SEQ ID	154	204	51	318	380	69	318	380	69
  gi|225377804|ref|ZP_03755025.1	NO: 347
  Methylosinus trichosporium OB3b	SEQ ID	144	193	50	426	488	64	426	488	64
  gi|296446027|ref|ZP_06887976.1	NO: 348
  Ruminococcus albus 8	SEQ ID	139	187	49	351	412	55	351	412	55
  gi|325677756|ref|ZP_08157403.1	NO: 349
  Bifidobacterium longum DJO10A	SEQ ID	183	230	48	370	431	44	370	431	44
  gi|189440764|ref|YP_001955845	NO: 350
  Enterococcus faecalis TX0012	SEQ ID	123	170	48	327	387	60	327	387	60
  gi|315149830|gb|EFT93846.1|	NO: 351
  Mycoplasma mobile 163K	SEQ ID	179	226	48	314	374	79	314	374	79
  gi|47458868|ref|YP_015730.1|	NO: 352
  Actinomyces coleocanis DSM 15436	SEQ ID	147	193	47	358	418	40	358	418	40
  gi|227494853|ref|ZP_03925169.1	NO: 353
  Dinoroseobacter shibae DFL 12	SEQ ID	138	184	47	338	398	48	338	398	48
  gi|159042956|ref|YP_001531750.1	NO: 354
  Actinomyces sp. oral taxon 180 str. F0310	SEQ ID		183	228	46	349	409	40	349	409	40
  gi|315605738|ref|ZP_07880770.1	NO: 355
  Alcanivorax sp. W11-5	SEQ ID	139	183	45	344	404	61	344	404	61
  gi|407803669|ref|ZP_11150502.1	NO: 356
  Aminomonas paucivorans DSM 12260	SEQ ID	134	178	45	341	401	63	341	401	63
  gi|312879015|ref|ZP_07738815.1	NO: 357
  Mycoplasma canis PG 14	SEQ ID	139	183	45	319	379	76	319	379	76
  gi|384393286|gb|EIE39736.1|	NO: 358
  Lactobacillus coryniformis KCTC 3535	SEQ ID	141	184	44	328	387	61	328	387	61
  gi|336393381|ref|ZP_08574780.1	NO: 359
  Elusimicrobium minutum Pei191	SEQ ID		177	219	43	322	381	47	322	381	47
  gi|187250660|ref|YP_001875142.1	NO: 360
  Neisseria meningitidis Z2491	SEQ ID		147	189	43	360	419	61	360	419	61
  gi|218767588|ref|YP_002342100.1	NO: 361
  Pasteurella multocida str. Pm70	SEQ ID		139	181	43	319	378	61	319	378	61
  gi|15602992|ref|NP_246064.1|	NO: 362
  Rhodovulum sp. PH10	SEQ ID		141	183	43	319	378	48	319	378	48
  gi|402849997|ref|ZP_10898214.1	NO: 363
  Eubacterium dolichum DSM 3991	SEQ ID	131	172	42	303	361	59	303	361	59
  gi|160915782|ref|ZP_02077990.1	NO: 364
  Nitratifractor salsuginis DSM 16511	SEQ ID	143	184	42	347	404	61	347	404	61
  gi|319957206|ref|YP_004168469.1	NO: 365
  Rhodospirillum rubrum ATCC 11170	SEQ ID	139	180	42	314	371	55	314	371	55
  gi|83591793|ref|YP_425545.1|	NO: 366
  Clostridium cellulolyticum H10	SEQ ID		137	176	40	320	376	61	320	376	61
  gi|220930482|ref|YP_002507391.1	NO: 367
  Helicobacter mustelae 12198	SEQ ID	148	187	40	298	354	48	298	354	48
  gi|291276265|ref|YP_003516037.1	NO: 368
  Ilyobacter polytropus DSM 2926	SEQ ID	134	173	40	462	517	63	462	517	63
  gi|310780384|ref|YP_003968716.1	NO: 369
  Sphaerochaeta globus str. Buddy	SEQ ID		163	202	40	335	389	45	335	389	45
  gi|325972003|ref|YP_004248194.1	NO: 370
  Staphylococcus lugdunensis M23590	SEQ ID		128	167	40	337	391	57	337	391	57
  gi|315659848|ref|ZP_07912707.1	NO: 371
  Treponema sp. JC4	SEQ ID		144	183	40	328	382	63	328	382	63
  gi|384109266|ref|ZP_10010146.1	NO: 372
  uncultured delta proteobacterium	SEQ ID		154	193	40	313	365	55	313	365	55
  HF0070 07E19	NO: 373
  gi|297182908|gb|ADI19058.1|
  Alicycliphilus denitrificans K601	SEQ ID		140	178	39	317	366	48	317	366	48
  gi|330822845|ref|YP_004386148.1	NO: 374
  Azospirillum sp. B510	SEQ ID		205	243	39	342	389	46	342	389	46
  gi|288957741|ref|YP_003448082.1	NO: 375
  Bradyrhizobium sp. BTAi1	SEQ ID		143	181	39	323	370	48	323	370	48
  gi|148255343|ref|YP_001239928.1	NO: 376
  Parvibaculum lavamentivorans DS-1	SEQ ID	138	176	39	327	374	58	327	374	58
  gi|154250555|ref|YP_001411379.1	NO: 377
  Prevotella timonensis CRIS 5C-B1	SEQ ID		170	208	39	328	375	61	328	375	61
  gi|282880052|ref|ZP_06288774.1	NO: 378
  Bacillus smithii 7 3 47FAA	SEQ ID		134	171	38	401	448	63	401	448	63
  gi|365156657|ref|ZP_09352959.1	NO: 379
  Cand. Puniceispirillum marinum IMCC1322	SEQ ID		135	172	38	344	391	53	344	391	53
  gi|294086111|ref|YP_003552871.1	NO: 380
  Barnesiella intestinihominis YIT 11860	SEQ ID	140	176	37	371	417	60	371	417	60
  gi|404487228|ref|ZP_11022414.1	NO: 381
  Ralstonia syzygii R24	SEQ ID		140	176	37	395	440	50	395	440	50
  gi|344171927|emb|CCA84553.1|	NO: 382
  Wolinella succinogenes DSM 1740	SEQ ID	145	180	36	348	392	60	348	392	60
  gi|34557790|ref|NP_907605.1|	NO: 383
  Mycoplasma gallisepticum str. F	SEQ ID		144	177	34	373	416	71	373	416	71
  gi|284931710|gb|ADC31648.1|	NO: 384
  Acidothermus cellulolyticus 11B	SEQ ID		150	182	33	341	380	58	341	380	58
  gi|117929158|ref|YP_873709.1|	NO: 385
  Mycoplasma ovipneumoniae SC01	SEQ ID		156	184	29	381	420	62	381	420	62
  gi|363542550|ref|ZP_09312133.1	NO: 386

• TABLE 9
  Amino Acid Sequence of Cas9 Core Domains

  Cas9 Start (AA pos)

  Cas9 Stop (AA pos)

  	Start and Stop numbers refer to the
  Strain Name	sequence in Table 7

  Staphylococcus Aureus	1	772
  Streptococcus Pyogenes	1	1099
  Campulobacter Jejuni	1	741

• TABLE 10
  Identified PAM sequences and
  corresponding RKR motifs

  		RKR
  	PAM sequence	motif
  Strain Name	(NA)	(AA)
  Streptococcus pyogenes	NGG	RKR
  Streptococcus mutans	NGG	RKR
  Streptococcus	NGGNG	RYR
  thermophilus A
  Treponema denticola	NAAAAN	VAK
  Streptococcus	NNAAAAW	IYK
  thermophilus B
  Campylobacter jejuni	NNNNACA	NLK
  Pasteurella multocida	GNNNCNNA	KDG
  Neisseria meningitidis	NNNNGATT or	IGK
  Staphylococcus aureus	NNGRRV (R = A or G;	NDK
  	V = A, G or C)
  	NNGRRT (R = A or G)

  
  PI domains are provided in Tables 11 and 12.

• TABLE 11
  Altered PI Domains

  	PI Start	PI Stop (AA
  	(AA pos)	pos)

  	Start and Stop numbers
  	refer to the sequences in	Length of PI	RKR
  Strain Name	Table 100	(AA)	motif (AA)

  Alicycliphilus	837	1029	193	--Y
  denitrificans
  K601
  Campylobacter	741	984	244	-NG
  jejuni NCTC
  11168
  Helicobacter	771	1024	254	-NQ
  mustelae 12198

• TABLE 12
  Other Altered PI Domains

  	PI Start	PI Stop (AA
  	(AA pos)	pos)

  	Start and Stop numbers
  	refer to the sequences in	Length of PI
  Strain Name	Table 7	(AA)	RKR motif (AA)

  Akkermansia muciniphila ATCC BAA-835	871	1101	231	ALK
  Ralstonia syzygii R24	821	1062	242	APY
  Cand. Puniceispirillum marinum IMCC1322	815	1035	221	AYK
  Fructobacillus fructosus KCTC 3544	1074	1323	250	DGN
  Eubacterium yurii ATCC 43715	1107	1391	285	DGY
  Eubacterium dolichum DSM 3991	779	1096	318	DKK
  Dinoroseobacter shibae DFL 12	851	1079	229	DPI
  Clostridium cellulolyticum H10	767	1021	255	EGK
  Pasteurella multocida str. Pm70	815	1056	242	ENN
  Mycoplasma canis PG 14	907	1233	327	EPK
  Porphyromonas sp. oral taxon 279 str. F0450	935	1197	263	EPT
  Filifactor alocis ATCC 35896	1094	1365	272	EVD
  Aminomonas paucivorans DSM 12260	801	1052	252	EVY
  Wolinella succinogenes DSM 1740	1034	1409	376	EYK
  Oenococcus kitaharae DSM 17330	1119	1389	271	GAL
  Coriobacterium glomerans PW2	1126	1384	259	GDR
  Peptoniphilus duerdenii ATCC BAA-1640	1091	1364	274	GDS
  Bifidobacterium bifidum S17	1138	1420	283	GGL
  Alicyclobacillus hesperidum URH17-3-68	876	1146	271	GGR
  Roseburia inulinivorans DSM 16841	895	1152	258	GGT
  Actinomyces coleocanis DSM 15436	843	1105	263	GKK
  Odoribacter laneus YIT 12061	1103	1498	396	GKV
  Coprococcus catus GD-7	1063	1338	276	GNQ
  Enterococcus faecalis TX0012	829	1150	322	GRK
  Bacillus smithii
  7 3 47FAA	809	1088	280	GSK
  Legionella pneumophila str. Paris	1021	1372	352	GTM
  Bacteroides fragilis NCTC 9343	1140	1436	297	IPV
  Mycoplasma ovipneumoniae SC01	923	1265	343	IRI
  Actinomyces sp. oral taxon 180 str. F0310	895	1181	287	KEK
  Treponema sp. JC4	832	1062	231	KIS
  Fusobacteriumnucleatum ATCC49256	1073	1374	302	KKV
  Lactobacillus farciminis KCTC 3681	1101	1356	256	KKV
  Nitratifractor salsuginis DSM 16511	840	1132	293	KMR
  Lactobacillus coryniformis KCTC 3535	850	1119	270	KNK
  Mycoplasma mobile 163K	916	1236	321	KNY
  Flavobacterium branchiophilum FL-15	1182	1473	292	KQK
  Prevotella timonensis CRIS 5C-B1	957	1218	262	KQQ
  Methylosinus trichosporium OB3b	830	1082	253	KRP
  Prevotella sp. C561	1099	1424	326	KRY
  Mycoplasma gallisepticum str. F	911	1269	359	KTA
  Lactobacillus rhamnosus GG	1077	1363	287	KYG
  Wolinella succinogenes DSM 1740	811	1059	249	LPN
  Streptococcus thermophilus LMD-9	1099	1388	290	MLA
  Treponema denticola ATCC 35405	1092	1395	304	NDS
  Bergeyella zoohelcum ATCC 43767	1098	1415	318	NEK
  Veillonella atypica ACS-134-V-Col7a	1107	1398	292	NGF
  Neisseria meningitidis Z2491	835	1082	248	NHN
  Ignavibacterium album JCM 16511	1296	1688	393	NKK
  Ruminococcus albus
  8	853	1156	304	NNF
  Streptococcus thermophilus LMD-9	811	1121	311	NNK
  Barnesiella intestinihominis YIT 11860	871	1153	283	NPV
  Azospirillum sp. B510	911	1168	258	PFH
  Rhodospirillum rubrum ATCC 11170	863	1173	311	PRG
  Planococcus antarcticus DSM 14505	1087	1333	247	PYY
  Staphylococcus pseudintermedius ED99	1073	1334	262	QIV
  Alcanivorax sp. W11-5	843	1113	271	RIE
  Bradyrhizobium sp. BTAi1	811	1064	254	RIY
  Streptococcus pyogenes M1 GAS	1099	1368	270	RKR
  Streptococcus mutans UA159	1078	1345	268	RKR
  Streptococcus Pyogenes
  	1099	1368	270	RKR
  Bacteroides sp. 20 3	1147	1517	371	RNI
  S. aureus
  	772	1053	282	RNK
  Solobacterium moorei F0204	1062	1327	266	RSG
  Finegoldia magna ATCC 29328	1081	1348	268	RTE
  uncultured delta proteobacterium HF0070 07E19	770	1011	242	SGG
  Acidaminococcus sp. D21	1064	1358	295	SIG
  Eubacterium rectale ATCC 33656	824	1114	291	SKK
  Caenispirillum salinarum AK4	1048	1442	395	SLV
  Acidothermus cellulolyticus 11B	830	1138	309	SPS
  Catenibacterium mitsuokai DSM 15897	1068	1329	262	SPT
  Parvibaculum lavamentivorans DS-1	827	1037	211	TGN
  Staphylococcus lugdunensis M23590	772	1054	283	TKK
  Streptococcus sanguinis SK49	1123	1421	299	TRM
  Elusimicrobium minutum Pei191	910	1195	286	TTG
  Nitrobacter hamburgensis X14	914	1166	253	VAY
  Mycoplasma synoviae 53	991	1314	324	VGF
  Sphaerochaeta globus str. Buddy	877	1179	303	VKG
  Ilyobacter polytropus DSM 2926	837	1092	256	VNG
  Rhodovulum sp. PH10	821	1059	239	VPY
  Bifidobacterium longum DJO10A	904	1187	284	VRK

Amino Acid Sequences Described in Table 8:

• SEQ ID NO: 304

  MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI
  QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDT
  GNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQ
  LDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY
  NALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK
  PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQIS
  NLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSP
  VVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTT
  GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVK
  QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD
  FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAED
  ALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKD
  YKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHH
  DPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDD
  YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA
  EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKT
  QSIKKYSTDILGNLYEVKSKKHPQIIKKG

  SEQ ID NO: 305

  MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL
  KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY
  HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY
  NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF
  DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS
  MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
  GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI
  PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS
  LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
  SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA
  HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF
  KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ
  TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR
  LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK
  FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS
  KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK
  SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
  MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
  KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS
  AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV
  ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
  ATLIHQSITGLYETRIDLSQLGGD

  SEQ ID NO: 306

  MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRKAR
  LNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKR
  RGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYE
  RCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAP
  KNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYE
  FKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDS
  LSKLEFKDHLNISFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVT
  NPVVLRAIKEYRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELEC
  EKLGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVL
  VFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDT
  RYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNH
  LHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLD
  KIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDMFR
  VDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILI
  QTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVF
  EKYIVSALGEVTKAEFRQREDFKK

  SEQ ID NO: 307

  MKRILGLDLGTNSIGWALVNEAENKDERSSIVKLGVRVNPLTVDELTNFEKGKSITTNADRTLK
  RGMRRNLQRYKLRRETLTEVLKEHKLITEDTILSENGNRTTFETYRLRAKAVTEEISLEEFARV
  LLMINKKRGYKSSRKAKGVEEGTLIDGMDIARELYNNNLTPGELCLQLLDAGKKFLPDFYRSDL
  QNELDRIWEKQKEYYPEILTDVLKEELRGKKRDAVWAICAKYFVWKENYTEWNKEKGKTEQQER
  EHKLEGIYSKRKRDEAKRENLQWRVNGLKEKLSLEQLVIVFQEMNTQINNSSGYLGAISDRSKE
  LYFNKQTVGQYQMEMLDKNPNASLRNMVFYRQDYLDEFNMLWEKQAVYHKELTEELKKEIRDII
  IFYQRRLKSQKGLIGFCEFESRQIEVDIDGKKKIKTVGNRVISRSSPLFQEFKIWQILNNIEVT
  VVGKKRKRRKLKENYSALFEELNDAEQLELNGSRRLCQEEKELLAQELFIRDKMTKSEVLKLLF
  DNPQELDLNFKTIDGNKTGYALFQAYSKMIEMSGHEPVDFKKPVEKVVEYIKAVFDLLNWNTDI
  LGFNSNEELDNQPYYKLWHLLYSFEGDNTPTGNGRLIQKMTELYGFEKEYATILANVSFQDDYG
  SLSAKAIHKILPHLKEGNRYDVACVYAGYRHSESSLTREEIANKVLKDRLMLLPKNSLHNPVVE
  KILNQMVNVINVIIDIYGKPDEIRVELARELKKNAKEREELTKSIAQTTKAHEEYKTLLQTEFG
  LTNVSRTDILRYKLYKELESCGYKTLYSNTYISREKLFSKEFDIEHIIPQARLFDDSFSNKTLE
  ARSVNIEKGNKTAYDFVKEKFGESGADNSLEHYLNNIEDLFKSGKISKTKYNKLKMAEQDIPDG
  FIERDLRNTQYIAKKALSMLNEISHRVVATSGSVTDKLREDWQLIDVMKELNWEKYKALGLVEY
  FEDRDGRQIGRIKDWTKRNDHRHHAMDALTVAFTKDVFIQYFNNKNASLDPNANEHAIKNKYFQ
  NGRAIAPMPLREFRAEAKKHLENTLISIKAKNKVITGNINKTRKKGGVNKNMQQTPRGQLHLET
  IYGSGKQYLTKEEKVNASFDMRKIGTVSKSAYRDALLKRLYENDNDPKKAFAGKNSLDKQPIWL
  DKEQMRKVPEKVKIVTLEAIYTIRKEISPDLKVDKVIDVGVRKILIDRLNEYGNDAKKAFSNLD
  KNPIWLNKEKGISIKRVTISGISNAQSLHVKKDKDGKPILDENGRNIPVDFVNTGNNHHVAVYY
  RPVIDKRGQLVVDEAGNPKYELEEVVVSFFEAVTRANLGLPIIDKDYKTTEGWQFLFSMKQNEY
  FVFPNEKTGFNPKEIDLLDVENYGLISPNLFRVQKFSLKNYVFRHHLETTIKDTSSILRGITWI
  DFRSSKGLDTIVKVRVNHIGQIVSVGEY

  SEQ ID NO: 308

  MSRKNYVDDYAISLDIGNASVGWSAFTPNYRLVRAKGHELIGVRLFDPADTAESRRMARTTRRR
  YSRRRWRLRLLDALFDQALSEIDPSFLARRKYSWVHPDDENNADCWYGSVLFDSNEQDKRFYEK
  YPTIYHLRKALMEDDSQHDIREIYLAIHHMVKYRGNFLVEGTLESSNAFKEDELLKLLGRITRY
  EMSEGEQNSDIEQDDENKLVAPANGQLADALCATRGSRSMRVDNALEALSAVNDLSREQRAIVK
  AIFAGLEGNKLDLAKIFVSKEFSSENKKILGIYFNKSDYEEKCVQIVDSGLLDDEEREFLDRMQ
  GQYNAIALKQLLGRSTSVSDSKCASYDAHRANWNLIKLQLRTKENEKDINENYGILVGWKIDSG
  QRKSVRGESAYENMRKKANVFFKKMIETSDLSETDKNRLIHDIEEDKLFPIQRDSDNGVIPHQL
  HQNELKQIIKKQGKYYPFLLDAFEKDGKQINKIEGLLTFRVPYFVGPLVVPEDLQKSDNSENHW
  MVRKKKGEITPWNFDEMVDKDASGRKFIERLVGTDSYLLGEPTLPKNSLLYQEYEVLNELNNVR
  LSVRTGNHWNDKRRMRLGREEKTLLCQRLFMKGQTVTKRTAENLLRKEYGRTYELSGLSDESKF
  TSSLSTYGKMCRIFGEKYVNEHRDLMEKIVELQTVFEDKETLLHQLRQLEGISEADCALLVNTH
  YTGWGRLSRKLLTTKAGECKISDDFAPRKHSIIEIMRAEDRNLMEIITDKQLGFSDWIEQENLG
  AENGSSLMEVVDDLRVSPKVKRGIIQSIRLIDDISKAVGKRPSRIFLELADDIQPSGRTISRKS
  RLQDLYRNANLGKEFKGIADELNACSDKDLQDDRLFLYYTQLGKDMYTGEELDLDRLSSAYDID
  HIIPQAVTQNDSIDNRVLVARAENARKTDSFTYMPQIADRMRNFWQILLDNGLISRVKFERLTR
  QNEFSEREKERFVQRSLVETRQIMKNVATLMRQRYGNSAAVIGLNAELTKEMHRYLGFSHKNRD
  INDYHHAQDALCVGIAGQFAANRGFFADGEVSDGAQNSYNQYLRDYLRGYREKLSAEDRKQGRA
  FGFIVGSMRSQDEQKRVNPRTGEVVWSEEDKDYLRKVMNYRKMLVTQKVGDDFGALYDETRYAA
  TDPKGIKGIPFDGAKQDTSLYGGFSSAKPAYAVLIESKGKTRLVNVTMQEYSLLGDRPSDDELR
  KVLAKKKSEYAKANILLRHVPKMQLIRYGGGLMVIKSAGELNNAQQLWLPYEEYCYFDDLSQGK
  GSLEKDDLKKLLDSILGSVQCLYPWHRFTEEELADLHVAFDKLPEDEKKNVITGIVSALHADAK
  TANLSIVGMTGSWRRMNNKSGYTFSDEDEFIFQSPSGLFEKRVTVGELKRKAKKEVNSKYRTNE
  KRLPTLSGASQP

  SEQ ID NO: 309

  METQTSNQLITSHLKDYPKQDYFVGLDIGTNSVGWAVTNTSYELLKFHSHKMWGSRLFEEGESA
  VTRRGFRSMRRRLERRKLRLKLLEELFADAMAQVDSTFFIRLHESKYHYEDKTTGHSSKHILFI
  DEDYTDQDYFTEYPTIYHLRKDLMENGTDDIRKLFLAVHHILKYRGNFLYEGATFNSNAFTFED
  VLKQALVNITFNCFDTNSAISSISNILMESGKTKSDKAKAIERLVDTYTVFDEVNTPDKPQKEQ
  VKEDKKTLKAFANLVLGLSANLIDLFGSVEDIDDDLKKLQIVGDTYDEKRDELAKVWGDEIHII
  DDCKSVYDAIILMSIKEPGLTISQSKVKAFDKHKEDLVILKSLLKLDRNVYNEMFKSDKKGLHN
  YVHYIKQGRTEETSCSREDFYKYTKKIVEGLADSKDKEYILNEIELQTLLPLQRIKDNGVIPYQ
  LHLEELKVILDKCGPKFPFLHTVSDGFSVTEKLIKMLEFRIPYYVGPLNTHHNIDNGGFSWAVR
  KQAGRVTPWNFEEKIDREKSAAAFIKNLTNKCTYLFGEDVLPKSSLLYSEFMLLNELNNVRIDG
  KALAQGVKQHLIDSIFKQDHKKMTKNRIELFLKDNNYITKKHKPEITGLDGEIKNDLTSYRDMV
  RILGNNFDVSMAEDIITDITIFGESKKMLRQTLRNKFGSQLNDETIKKLSKLRYRDWGRLSKKL
  LKGIDGCDKAGNGAPKTIIELMRNDSYNLMEILGDKFSFMECIEEENAKLAQGQVVNPHDIIDE
  LALSPAVKRAVWQALRIVDEVAHIKKALPSRIFVEVARTNKSEKKKKDSRQKRLSDLYSAIKKD
  DVLQSGLQDKEFGALKSGLANYDDAALRSKKLYLYYTQMGRCAYTGNIIDLNQLNTDNYDIDHI
  YPRSLTKDDSFDNLVLCERTANAKKSDIYPIDNRIQTKQKPFWAFLKHQGLISERKYERLTRIA
  PLTADDLSGFIARQLVETNQSVKATTTLLRRLYPDIDVVFVKAENVSDFRHNNNFIKVRSLNHH
  HHAKDAYLNIVVGNVYHEKFTRNFRLFFKKNGANRTYNLAKMFNYDVICTNAQDGKAWDVKTSM
  NTVKKMMASNDVRVTRRLLEQSGALADATIYKASVAAKAKDGAYIGMKTKYSVFADVTKYGGMT
  KIKNAYSIIVQYTGKKGEEIKEIVPLPIYLINRNATDIELIDYVKSVIPKAKDISIKYRKLCIN
  QLVKVNGFYYYLGGKTNDKIYIDNAIELVVPHDIATYIKLLDKYDLLRKENKTLKASSITTSIY
  NINTSTVVSLNKVGIDVFDYFMSKLRTPLYMKMKGNKVDELSSTGRSKFIKMTLEEQSIYLLEV
  LNLLTNSKTTFDVKPLGITGSRSTIGVKIHNLDEFKIINESITGLYSNEVTIV

  SEQ ID NO: 310

  MTKLNQPYGIGLDIGSNSIGFAVVDANSHLLRLKGETAIGARLFREGQSAADRRGSRTTRRRLS
  RTRWRLSFLRDFFAPHITKIDPDFFLRQKYSEISPKDKDRFKYEKRLFNDRTDAEFYEDYPSMY
  HLRLHLMTHTHKADPREIFLAIHHILKSRGHFLTPGAAKDFNTDKVDLEDIFPALTEAYAQVYP
  DLELTFDLAKADDFKAKLLDEQATPSDTQKALVNLLLSSDGEKEIVKKRKQVLTEFAKAITGLK
  TKFNLALGTEVDEADASNWQFSMGQLDDKWSNIETSMTDQGTEIFEQIQELYRARLLNGIVPAG
  MSLSQAKVADYGQHKEDLELFKTYLKKLNDHELAKTIRGLYDRYINGDDAKPFLREDFVKALTK
  EVTAHPNEVSEQLLNRMGQANFMLKQRTKANGAIPIQLQQRELDQIIANQSKYYDWLAAPNPVE
  AHRWKMPYQLDELLNFHIPYYVGPLITPKQQAESGENVFAWMVRKDPSGNITPYNFDEKVDREA
  SANTFIQRMKTTDTYLIGEDVLPKQSLLYQKYEVLNELNNVRINNECLGTDQKQRLIREVFERH
  SSVTIKQVADNLVAHGDFARRPEIRGLADEKRFLSSLSTYHQLKEILHEAIDDPTKLLDIENII
  TWSTVFEDHTIFETKLAEIEWLDPKKINELSGIRYRGWGQFSRKLLDGLKLGNGHTVIQELMLS
  NHNLMQILADETLKETMTELNQDKLKTDDIEDVINDAYTSPSNKKALRQVLRVVEDIKHAANGQ
  DPSWLFIETADGTGTAGKRTQSRQKQIQTVYANAAQELIDSAVRGELEDKIADKASFTDRLVLY
  FMQGGRDIYTGAPLNIDQLSHYDIDHILPQSLIKDDSLDNRVLVNATINREKNNVFASTLFAGK
  MKATWRKWHEAGLISGRKLRNLMLRPDEIDKFAKGFVARQLVETRQIIKLTEQIAAAQYPNTKI
  IAVKAGLSHQLREELDFPKNRDVNHYHHAFDAFLAARIGTYLLKRYPKLAPFFTYGEFAKVDVK
  KFREFNFIGALTHAKKNIIAKDTGEIVWDKERDIRELDRIYNFKRMLITHEVYFETADLFKQTI
  YAAKDSKERGGSKQLIPKKQGYPTQVYGGYTQESGSYNALVRVAEADTTAYQVIKISAQNASKI
  ASANLKSREKGKQLLNEIVVKQLAKRRKNWKPSANSFKIVIPRFGMGTLFQNAKYGLFMVNSDT
  YYRNYQELWLSRENQKLLKKLFSIKYEKTQMNHDALQVYKAIIDQVEKFFKLYDINQFRAKLSD
  AIERFEKLPINTDGNKIGKTETLRQILIGLQANGTRSNVKNLGIKTDLGLLQVGSGIKLDKDTQ
  IVYQSPSGLFKRRIPLADL

  SEQ ID NO: 311

  MTKEYYLGLDVGTNSVGWAVTDSQYNLCKFKKKDMWGIRLFESANTAKDRRLQRGNRRRLERKK
  QRIDLLQEIFSPEICKIDPTFFIRLNESRLHLEDKSNDFKYPLFIEKDYSDIEYYKEFPTIFHL
  RKHLIESEEKQDIRLIYLALHNIIKTRGHFLIDGDLQSAKQLRPILDTFLLSLQEEQNLSVSLS
  ENQKDEYEEILKNRSIAKSEKVKKLKNLFEISDELEKEEKKAQSAVIENFCKFIVGNKGDVCKF
  LRVSKEELEIDSFSFSEGKYEDDIVKNLEEKVPEKVYLFEQMKAMYDWNILVDILETEEYISFA
  KVKQYEKHKTNLRLLRDIILKYCTKDEYNRMFNDEKEAGSYTAYVGKLKKNNKKYWIEKKRNPE
  EFYKSLGKLLDKIEPLKEDLEVLTMMIEECKNHTLLPIQKNKDNGVIPHQVHEVELKKILENAK
  KYYSFLTETDKDGYSVVQKIESIFRFRIPYYVGPLSTRHQEKGSNVWMVRKPGREDRIYPWNME
  EIIDFEKSNENFITRMTNKCTYLIGEDVLPKHSLLYSKYMVLNELNNVKVRGKKLPTSLKQKVF
  EDLFENKSKVTGKNLLEYLQIQDKDIQIDDLSGFDKDFKTSLKSYLDFKKQIFGEEIEKESIQN
  MIEDIIKWITIYGNDKEMLKRVIRANYSNQLTEEQMKKITGFQYSGWGNFSKMFLKGISGSDVS
  TGETFDIITAMWETDNNLMQILSKKFTFMDNVEDFNSGKVGKIDKITYDSTVKEMFLSPENKRA
  VWQTIQVAEEIKKVMGCEPKKIFIEMARGGEKVKKRTKSRKAQLLELYAACEEDCRELIKEIED
  RDERDFNSMKLFLYYTQFGKCMYSGDDIDINELIRGNSKWDRDHIYPQSKIKDDSIDNLVLVNK
  TYNAKKSNELLSEDIQKKMHSFWLSLLNKKLITKSKYDRLTRKGDFTDEELSGFIARQLVETRQ
  STKAIADIFKQIYSSEVVYVKSSLVSDFRKKPLNYLKSRRVNDYHHAKDAYLNIVVGNVYNKKF
  TSNPIQWMKKNRDTNYSLNKVFEHDVVINGEVIWEKCTYHEDTNTYDGGTLDRIRKIVERDNIL
  YTEYAYCEKGELFNATIQNKNGNSTVSLKKGLDVKKYGGYFSANTSYFSLIEFEDKKGDRARHI
  IGVPIYIANMLEHSPSAFLEYCEQKGYQNVRILVEKIKKNSLLIINGYPLRIRGENEVDTSFKR
  AIQLKLDQKNYELVRNIEKFLEKYVEKKGNYPIDENRDHITHEKMNQLYEVLLSKMKKFNKKGM
  ADPSDRIEKSKPKFIKLEDLIDKINVINKMLNLLRCDNDTKADLSLIELPKNAGSFVVKKNTIG
  KSKIILVNQSVTGLYENRREL

  SEQ ID NO: 312

  MARDYSVGLDIGTSSVGWAAIDNKYHLIRAKSKNLIGVRLFDSAVTAEKRRGYRTTRRRLSRRH
  WRLRLLNDIFAGPLTDFGDENFLARLKYSWVHPQDQSNQAHFAAGLLFDSKEQDKDFYRKYPTI
  YHLRLALMNDDQKHDLREVYLAIHHLVKYRGHFLIEGDVKADSAFDVHTFADAIQRYAESNNSD
  ENLLGKIDEKKLSAALTDKHGSKSQRAETAETAFDILDLQSKKQIQAILKSVVGNQANLMAIFG
  LDSSAISKDEQKNYKFSFDDADIDEKIADSEALLSDTEFEFLCDLKAAFDGLTLKMLLGDDKTV
  SAAMVRRFNEHQKDWEYIKSHIRNAKNAGNGLYEKSKKFDGINAAYLALQSDNEDDRKKAKKIF
  QDEISSADIPDDVKADFLKKIDDDQFLPIQRTKNNGTIPHQLHRNELEQIIEKQGIYYPFLKDT
  YQENSHELNKITALINFRVPYYVGPLVEEEQKIADDGKNIPDPTNHWMVRKSNDTITPWNLSQV
  VDLDKSGRRFIERLTGTDTYLIGEPTLPKNSLLYQKFDVLQELNNIRVSGRRLDIRAKQDAFEH
  LFKVQKTVSATNLKDFLVQAGYISEDTQIEGLADVNGKNFNNALTTYNYLVSVLGREFVENPSN
  EELLEEITELQTVFEDKKVLRRQLDQLDGLSDHNREKLSRKHYTGWGRISKKLLTTKIVQNADK
  IDNQTFDVPRMNQSIIDTLYNTKMNLMEIINNAEDDFGVRAWIDKQNTTDGDEQDVYSLIDELA
  GPKEIKRGIVQSFRILDDITKAVGYAPKRVYLEFARKTQESHLTNSRKNQLSTLLKNAGLSELV
  TQVSQYDAAALQNDRLYLYFLQQGKDMYSGEKLNLDNLSNYDIDHIIPQAYTKDNSLDNRVLVS
  NITNRRKSDSSNYLPALIDKMRPFWSVLSKQGLLSKHKFANLTRTRDFDDMEKERFIARSLVET
  RQIIKNVASLIDSHFGGETKAVAIRSSLTADMRRYVDIPKNRDINDYHHAFDALLFSTVGQYTE
  NSGLMKKGQLSDSAGNQYNRYIKEWIHAARLNAQSQRVNPFGFVVGSMRNAAPGKLNPETGEIT
  PEENADWSIADLDYLHKVMNFRKITVTRRLKDQKGQLYDESRYPSVLHDAKSKASINFDKHKPV
  DLYGGFSSAKPAYAALIKFKNKFRLVNVLRQWTYSDKNSEDYILEQIRGKYPKAEMVLSHIPYG
  QLVKKDGALVTISSATELHNFEQLWLPLADYKLINTLLKTKEDNLVDILHNRLDLPEMTIESAF
  YKAFDSILSFAFNRYALHQNALVKLQAHRDDFNALNYEDKQQTLERILDALHASPASSDLKKIN
  LSSGFGRLFSPSHFTLADTDEFIFQSVTGLFSTQKTVAQLYQETK

  SEQ ID NO: 313

  MVYDVGLDIGTGSVGWVALDENGKLARAKGKNLVGVRLFDTAQTAADRRGFRTTRRRLSRRKWR
  LRLLDELFSAEINEIDSSFFQRLKYSYVHPKDEENKAHYYGGYLFPTEEETKKFHRSYPTIYHL
  RQELMAQPNKRFDIREIYLAIHHLVKYRGHFLSSQEKITIGSTYNPEDLANAIEVYADEKGLSW
  ELNNPEQLTEIISGEAGYGLNKSMKADEALKLFEFDNNQDKVAIKTLLAGLTGNQIDFAKLFGK
  DISDKDEAKLWKLKLDDEALEEKSQTILSQLTDEEIELFHAVVQAYDGFVLIGLLNGADSVSAA
  MVQLYDQHREDRKLLKSLAQKAGLKHKRFSEIYEQLALATDEATIKNGISTARELVEESNLSKE
  VKEDTLRRLDENEFLPKQRTKANSVIPHQLHLAELQKILQNQGQYYPFLLDTFEKEDGQDNKIE
  ELLRFRIPYYVGPLVTKKDVEHAGGDADNHWVERNEGFEKSRVTPWNFDKVFNRDKAARDFIER
  LTGNDTYLIGEKTLPQNSLRYQLFTVLNELNNVRVNGKKFDSKTKADLINDLFKARKTVSLSAL
  KDYLKAQGKGDVTITGLADESKFNSSLSSYNDLKKTFDAEYLENEDNQETLEKIIEIQTVFEDS
  KIASRELSKLPLDDDQVKKLSQTHYTGWGRLSEKLLDSKIIDERGQKVSILDKLKSTSQNFMSI
  INNDKYGVQAWITEQNTGSSKLTFDEKVNELTTSPANKRGIKQSFAVLNDIKKAMKEEPRRVYL
  EFAREDQTSVRSVPRYNQLKEKYQSKSLSEEAKVLKKTLDGNKNKMSDDRYFLYFQQQGKDMYT
  GRPINFERLSQDYDIDHIIPQAFTKDDSLDNRVLVSRPENARKSDSFAYTDEVQKQDGSLWTSL
  LKSGFINRKKYERLTKAGKYLDGQKTGFIARQLVETRQIIKNVASLIEGEYENSKAVAIRSEIT
  ADMRLLVGIKKHREINSFHHAFDALLITAAGQYMQNRYPDRDSTNVYNEFDRYTNDYLKNLRQL
  SSRDEVRRLKSFGFVVGTMRKGNEDWSEENTSYLRKVMMFKNILTTKKTEKDRGPLNKETIFSP
  KSGKKLIPLNSKRSDTALYGGYSNVYSAYMTLVRANGKNLLIKIPISIANQIEVGNLKINDYIV
  NNPAIKKFEKILISKLPLGQLVNEDGNLIYLASNEYRHNAKQLWLSTTDADKIASISENSSDEE
  LLEAYDILTSENVKNRFPFFKKDIDKLSQVRDEFLDSDKRIAVIQTILRGLQIDAAYQAPVKII
  SKKVSDWHKLQQSGGIKLSDNSEMIYQSATGIFETRVKISDLL

  SEQ ID NO: 314

  IVDYCIGLDLGTGSVGWAVVDMNHRLMKRNGKHLWGSRLFSNAETAANRRASRSIRRRYNKRRE
  RIRLLRAILQDMVLEKDPTFFIRLEHTSFLDEEDKAKYLGTDYKDNYNLFIDEDFNDYTYYHKY
  PTIYHLRKALCESTEKADPRLIYLALHHIVKYRGNFLYEGQKFNMDASNIEDKLSDIFTQFTSF
  NNIPYEDDEKKNLEILEILKKPLSKKAKVDEVMTLIAPEKDYKSAFKELVTGIAGNKMNVTKMI
  LCEPIKQGDSEIKLKFSDSNYDDQFSEVEKDLGEYVEFVDALHNVYSWVELQTIMGATHTDNAS
  ISEAMVSRYNKHHDDLKLLKDCIKNNVPNKYFDMFRNDSEKSKGYYNYINRPSKAPVDEFYKYV
  KKCIEKVDTPEAKQILNDIELENFLLKQNSRTNGSVPYQMQLDEMIKIIDNQAEYYPILKEKRE
  QLLSILTFRIPYYFGPLNETSEHAWIKRLEGKENQRILPWNYQDIVDVDATAEGFIKRMRSYCT
  YFPDEEVLPKNSLIVSKYEVYNELNKIRVDDKLLEVDVKNDIYNELFMKNKTVTEKKLKNWLVN
  NQCCSKDAEIKGFQKENQFSTSLTPWIDFTNIFGKIDQSNFDLIENIIYDLTVFEDKKIMKRRL
  KKKYALPDDKVKQILKLKYKDWSRLSKKLLDGIVADNRFGSSVTVLDVLEMSRLNLMEIINDKD
  LGYAQMIEEATSCPEDGKFTYEEVERLAGSPALKRGIWQSLQIVEEITKVMKCRPKYIYIEFER
  SEEAKERTESKIKKLENVYKDLDEQTKKEYKSVLEELKGFDNTKKISSDSLFLYFTQLGKCMYS
  GKKLDIDSLDKYQIDHIVPQSLVKDDSFDNRVLVVPSENQRKLDDLVVPFDIRDKMYRFWKLLF
  DHELISPKKFYSLIKTEYTERDEERFINRQLVETRQITKNVTQIIEDHYSTTKVAAIRANLSHE
  FRVKNHIYKNRDINDYHHAHDAYIVALIGGFMRDRYPNMHDSKAVYSEYMKMFRKNKNDQKRWK
  DGFVINSMNYPYEVDGKLIWNPDLINEIKKCFYYKDCYCTTKLDQKSGQLFNLTVLSNDAHADK
  GVTKAVVPVNKNRSDVHKYGGFSGLQYTIVAIEGQKKKGKKTELVKKISGVPLHLKAASINEKI
  NYIEEKEGLSDVRIIKDNIPVNQMIEMDGGEYLLTSPTEYVNARQLVLNEKQCALIADIYNAIY
  KQDYDNLDDILMIQLYIELTNKMKVLYPAYRGIAEKFESMNENYVVISKEEKANIIKQMLIVMH
  RGPQNGNIVYDDFKISDRIGRLKTKNHNLNNIVFISQSPTGIYTKKYKL

  SEQ ID NO: 315

  MKSEKKYYIGLDVGTNSVGWAVTDEFYNILRAKGKDLWGVRLFEKADTAANTRIFRSGRRRNDR
  KGMRLQILREIFEDEIKKVDKDFYDRLDESKFWAEDKKVSGKYSLFNDKNFSDKQYFEKFPTIF
  HLRKYLMEEHGKVDIRYYFLAINQMMKRRGHFLIDGQISHVTDDKPLKEQLILLINDLLKIELE
  EELMDSIFEILADVNEKRTDKKNNLKELIKGQDFNKQEGNILNSIFESIVTGKAKIKNIISDED
  ILEKIKEDNKEDFVLTGDSYEENLQYFEEVLQENITLFNTLKSTYDFLILQSILKGKSTLSDAQ
  VERYDEHKKDLEILKKVIKKYDEDGKLFKQVFKEDNGNGYVSYIGYYLNKNKKITAKKKISNIE
  FTKYVKGILEKQCDCEDEDVKYLLGKIEQENFLLKQISSINSVIPHQIHLFELDKILENLAKNY
  PSFNNKKEEFTKIEKIRKTFTFRIPYYVGPLNDYHKNNGGNAWIFRNKGEKIRPWNFEKIVDLH
  KSEEEFIKRMLNQCTYLPEETVLPKSSILYSEYMVLNELNNLRINGKPLDTDVKLKLIEELFKK
  KTKVTLKSIRDYMVRNNFADKEDFDNSEKNLEIASNMKSYIDFNNILEDKFDVEMVEDLIEKIT
  IHTGNKKLLKKYIEETYPDLSSSQIQKIINLKYKDWGRLSRKLLDGIKGTKKETEKTDTVINFL
  RNSSDNLMQIIGSQNYSFNEYIDKLRKKYIPQEISYEVVENLYVSPSVKKMIWQVIRVTEEITK
  VMGYDPDKIFIEMAKSEEEKKTTISRKNKLLDLYKAIKKDERDSQYEKLLTGLNKLDDSDLRSR
  KLYLYYTQMGRDMYTGEKIDLDKLFDSTHYDKDHIIPQSMKKDDSIINNLVLVNKNANQTTKGN
  IYPVPSSIRNNPKIYNYWKYLMEKEFISKEKYNRLIRNTPLTNEELGGFINRQLVETRQSTKAI
  KELFEKFYQKSKIIPVKASLASDLRKDMNTLKSREVNDLHHAHDAFLNIVAGDVWNREFTSNPI
  NYVKENREGDKVKYSLSKDFTRPRKSKGKVIWTPEKGRKLIVDTLNKPSVLISNESHVKKGELF
  NATIAGKKDYKKGKIYLPLKKDDRLQDVSKYGGYKAINGAFFFLVEHTKSKKRIRSIELFPLHL
  LSKFYEDKNTVLDYAINVLQLQDPKIIIDKINYRTEIIIDNFSYLISTKSNDGSITVKPNEQMY
  WRVDEISNLKKIENKYKKDAILTEEDRKIMESYIDKIYQQFKAGKYKNRRTTDTIIEKYEIIDL
  DTLDNKQLYQLLVAFISLSYKTSNNAVDFTVIGLGTECGKPRITNLPDNTYLVYKSITGIYEKR
  IRIK

  SEQ ID NO: 316

  MKLRGIEDDYSIGLDMGTSSVGWAVTDERGTLAHFKRKPTWGSRLFREAQTAAVARMPRGQRRR
  YVRRRWRLDLLQKLFEQQMEQADPDFFIRLRQSRLLRDDRAEEHADYRWPLFNDCKFTERDYYQ
  RFPTIYHVRSWLMETDEQADIRLIYLALHNIVKHRGNFLREGQSLSAKSARPDEALNHLRETLR
  VWSSERGFECSIADNGSILAMLTHPDLSPSDRRKKIAPLFDVKSDDAAADKKLGIALAGAVIGL
  KTEFKNIFGDFPCEDSSIYLSNDEAVDAVRSACPDDCAELFDRLCEVYSAYVLQGLLSYAPGQT
  ISANMVEKYRRYGEDLALLKKLVKIYAPDQYRMFFSGATYPGTGIYDAAQARGYTKYNLGPKKS
  EYKPSESMQYDDFRKAVEKLFAKTDARADERYRMMMDRFDKQQFLRRLKTSDNGSIYHQLHLEE
  LKAIVENQGRFYPFLKRDADKLVSLVSFRIPYYVGPLSTRNARTDQHGENRFAWSERKPGMQDE
  PIFPWNWESIIDRSKSAEKFILRMTGMCTYLQQEPVLPKSSLLYEEFCVLNELNGAHWSIDGDD
  EHRFDAADREGIIEELFRRKRTVSYGDVAGWMERERNQIGAHVCGGQGEKGFESKLGSYIFFCK
  DVFKVERLEQSDYPMIERIILWNTLFEDRKILSQRLKEEYGSRLSAEQIKTICKKRFTGWGRLS
  EKFLTGITVQVDEDSVSIMDVLREGCPVSGKRGRAMVMMEILRDEELGFQKKVDDFNRAFFAEN
  AQALGVNELPGSPAVRRSLNQSIRIVDEIASIAGKAPANIFIEVTRDEDPKKKGRRTKRRYNDL
  KDALEAFKKEDPELWRELCETAPNDMDERLSLYFMQRGKCLYSGRAIDIHQLSNAGIYEVDHII
  PRTYVKDDSLENKALVYREENQRKTDMLLIDPEIRRRMSGYWRMLHEAKLIGDKKFRNLLRSRI
  DDKALKGFIARQLVETGQMVKLVRSLLEARYPETNIISVKASISHDLRTAAELVKCREANDFHH
  AHDAFLACRVGLFIQKRHPCVYENPIGLSQVVRNYVRQQADIFKRCRTIPGSSGFIVNSFMTSG
  FDKETGEIFKDDWDAEAEVEGIRRSLNFRQCFISRMPFEDHGVFWDATIYSPRAKKTAALPLKQ
  GLNPSRYGSFSREQFAYFFIYKARNPRKEQTLFEFAQVPVRLSAQIRQDENALERYARELAKDQ
  GLEFIRIERSKILKNQLIEIDGDRLCITGKEEVRNACELAFAQDEMRVIRMLVSEKPVSRECVI
  SLFNRILLHGDQASRRLSKQLKLALLSEAFSEASDNVQRNVVLGLIAIFNGSTNMVNLSDIGGS
  KFAGNVRIKYKKELASPKVNVHLIDQSVTGMFERRTKIGL

  SEQ ID NO: 317

  MENKQYYIGLDVGTNSVGWAVTDTSYNLLRAKGKDMWGARLFEKANTAAERRTKRTSRRRSERE
  KARKAMLKELFADEINRVDPSFFIRLEESKFFLDDRSENNRQRYTLFNDATFTDKDYYEKYKTI
  FHLRSALINSDEKFDVRLVFLAILNLFSHRGHFLNASLKGDGDIQGMDVFYNDLVESCEYFEIE
  LPRITNIDNFEKILSQKGKSRTKILEELSEELSISKKDKSKYNLIKLISGLEASVVELYNIEDI
  QDENKKIKIGFRESDYEESSLKVKEIIGDEYFDLVERAKSVHDMGLLSNIIGNSKYLCEARVEA
  YENHHKDLLKIKELLKKYDKKAYNDMFRKMTDKNYSAYVGSVNSNIAKERRSVDKRKIEDLYKY
  IEDTALKNIPDDNKDKIEILEKIKLGEFLKKQLTASNGVIPNQLQSRELRAILKKAENYLPFLK
  EKGEKNLTVSEMIIQLFEFQIPYYVGPLDKNPKKDNKANSWAKIKQGGRILPWNFEDKVDVKGS
  RKEFIEKMVRKCTYISDEHTLPKQSLLYEKFMVLNEINNIKIDGEKISVEAKQKIYNDLFVKGK
  KVSQKDIKKELISLNIMDKDSVLSGTDTVCNAYLSSIGKFTGVFKEEINKQSIVDMIEDIIFLK
  TVYGDEKRFVKEEIVEKYGDEIDKDKIKRILGFKFSNWGNLSKSFLELEGADVGTGEVRSIIQS
  LWETNFNLMELLSSRFTYMDELEKRVKKLEKPLSEWTIEDLDDMYLSSPVKRMIWQSMKIVDEI
  QTVIGYAPKRIFVEMTRSEGEKVRTKSRKDRLKELYNGIKEDSKQWVKELDSKDESYFRSKKMY
  LYYLQKGRCMYSGEVIELDKLMDDNLYDIDHIYPRSFVKDDSLDNLVLVKKEINNRKQNDPITP
  QIQASCQGFWKILHDQGFMSNEKYSRLTRKTQEFSDEEKLSFINRQIVETGQATKCMAQILQKS
  MGEDVDVVFSKARLVSEFRHKFELFKSRLINDFHHANDAYLNIVVGNSYFVKFTRNPANFIKDA
  RKNPDNPVYKYHMDRFFERDVKSKSEVAWIGQSEGNSGTIVIVKKTMAKNSPLITKKVEEGHGS
  ITKETIVGVKEIKFGRNKVEKADKTPKKPNLQAYRPIKTSDERLCNILRYGGRTSISISGYCLV
  EYVKKRKTIRSLEAIPVYLGRKDSLSEEKLLNYFRYNLNDGGKDSVSDIRLCLPFISTNSLVKI
  DGYLYYLGGKNDDRIQLYNAYQLKMKKEEVEYIRKIEKAVSMSKFDEIDREKNPVLTEEKNIEL
  YNKIQDKFENTVFSKRMSLVKYNKKDLSFGDFLKNKKSKFEEIDLEKQCKVLYNIIFNLSNLKE
  VDLSDIGGSKSTGKCRCKKNITNYKEFKLIQQSITGLYSCEKDLMTI

  SEQ ID NO: 318

  MKNLKEYYIGLDIGTASVGWAVTDESYNIPKFNGKKMWGVRLFDDAKTAEERRTQRGSRRRLNR
  RKERINLLQDLFATEISKVDPNFFLRLDNSDLYREDKDEKLKSKYTLFNDKDFKDRDYHKKYPT
  IHHLIMDLIEDEGKKDIRLLYLACHYLLKNRGHFIFEGQKFDTKNSFDKSINDLKIHLRDEYNI
  DLEFNNEDLIEIITDTTLNKTNKKKELKNIVGDTKFLKAISAIMIGSSQKLVDLFEDGEFEETT
  VKSVDFSTTAFDDKYSEYEEALGDTISLLNILKSIYDSSILENLLKDADKSKDGNKYISKAFVK
  KFNKHGKDLKTLKRIIKKYLPSEYANIFRNKSINDNYVAYTKSNITSNKRTKASKFTKQEDFYK
  FIKKHLDTIKETKLNSSENEDLKLIDEMLTDIEFKTFIPKLKSSDNGVIPYQLKLMELKKILDN
  QSKYYDFLNESDEYGTVKDKVESIMEFRIPYYVGPLNPDSKYAWIKRENTKITPWNFKDIVDLD
  SSREEFIDRLIGRCTYLKEEKVLPKASLIYNEFMVLNELNNLKLNEFLITEEMKKAIFEELFKT
  KKKVTLKAVSNLLKKEFNLTGDILLSGTDGDFKQGLNSYIDFKNIIGDKVDRDDYRIKIEEIIK
  LIVLYEDDKTYLKKKIKSAYKNDFTDDEIKKIAALNYKDWGRLSKRFLTGIEGVDKTTGEKGSI
  IYFMREYNLNLMELMSGHYTFTEEVEKLNPVENRELCYEMVDELYLSPSVKRMLWQSLRVVDEI
  KRIIGKDPKKIFIEMARAKEAKNSRKESRKNKLLEFYKFGKKAFINEIGEERYNYLLNEINSEE
  ESKFRWDNLYLYYTQLGRCMYSLEPIDLADLKSNNIYDQDHIYPKSKIYDDSLENRVLVKKNLN
  HEKGNQYPIPEKVLNKNAYGFWKILFDKGLIGQKKYTRLTRRTPFEERELAEFIERQIVETRQA
  TKETANLLKNICQDSEIVYSKAENASRFRQEFDIIKCRTVNDLHHMHDAYLNIVVGNVYNTKFT
  KNPLNFIKDKDNVRSYNLENMFKYDVVRGSYTAWIADDSEGNVKAATIKKVKRELEGKNYRFTR
  MSYIGTGGLYDQNLMRKGKGQIPQKENTNKSNIEKYGGYNKASSAYFALIESDGKAGRERTLET
  IPIMVYNQEKYGNTEAVDKYLKDNLELQDPKILKDKIKINSLIKLDGFLYNIKGKTGDSLSIAG
  SVQLIVNKEEQKLIKKMDKFLVKKKDNKDIKVTSFDNIKEEELIKLYKTLSDKLNNGIYSNKRN
  NQAKNISEALDKFKEISIEEKIDVLNQIILLFQSYNNGCNLKSIGLSAKTGVVFIPKKLNYKEC
  KLINQSITGLFENEVDLLNL

  SEQ ID NO: 319

  MGKMYYLGLDIGTNSVGYAVTDPSYHLLKFKGEPMWGAHVFAAGNQSAERRSFRTSRRRLDRRQ
  QRVKLVQEIFAPVISPIDPRFFIRLHESALWRDDVAETDKHIFFNDPTYTDKEYYSDYPTIHHL
  IVDLMESSEKHDPRLVYLAVAWLVAHRGHFLNEVDKDNIGDVLSFDAFYPEFLAFLSDNGVSPW
  VCESKALQATLLSRNSVNDKYKALKSLIFGSQKPEDNFDANISEDGLIQLLAGKKVKVNKLFPQ
  ESNDASFTLNDKEDAIEEILGTLTPDECEWIAHIRRLFDWAIMKHALKDGRTISESKVKLYEQH
  HHDLTQLKYFVKTYLAKEYDDIFRNVDSETTKNYVAYSYHVKEVKGTLPKNKATQEEFCKYVLG
  KVKNIECSEADKVDFDEMIQRLTDNSFMPKQVSGENRVIPYQLYYYELKTILNKAASYLPFLTQ
  CGKDAISNQDKLLSIMTFRIPYFVGPLRKDNSEHAWLERKAGKIYPWNFNDKVDLDKSEEAFIR
  RMTNTCTYYPGEDVLPLDSLIYEKFMILNEINNIRIDGYPISVDVKQQVFGLFEKKRRVTVKDI
  QNLLLSLGALDKHGKLTGIDTTIHSNYNTYHHFKSLMERGVLTRDDVERIVERMTYSDDTKRVR
  LWLNNNYGTLTADDVKHISRLRKHDFGRLSKMFLTGLKGVHKETGERASILDFMWNTNDNLMQL
  LSECYTFSDEITKLQEAYYAKAQLSLNDFLDSMYISNAVKRPIYRTLAVVNDIRKACGTAPKRI
  FIEMARDGESKKKRSVTRREQIKNLYRSIRKDFQQEVDFLEKILENKSDGQLQSDALYLYFAQL
  GRDMYTGDPIKLEHIKDQSFYNIDHIYPQSMVKDDSLDNKVLVQSEINGEKSSRYPLDAAIRNK
  MKPLWDAYYNHGLISLKKYQRLTRSTPFTDDEKWDFINRQLVETRQSTKALAILLKRKFPDTEI
  VYSKAGLSSDFRHEFGLVKSRNINDLHHAKDAFLAIVTGNVYHERFNRRWFMVNQPYSVKTKTL
  FTHSIKNGNFVAWNGEEDLGRIVKMLKQNKNTIHFTRFSFDRKEGLFDIQPLKASTGLVPRKAG
  LDVVKYGGYDKSTAAYYLLVRFTLEDKKTQHKLMMIPVEGLYKARIDHDKEFLTDYAQTTISEI
  LQKDKQKVINIMFPMGTRHIKLNSMISIDGFYLSIGGKSSKGKSVLCHAMVPLIVPHKIECYIK
  AMESFARKFKENNKLRIVEKFDKITVEDNLNLYELFLQKLQHNPYNKFFSTQFDVLTNGRSTFT
  KLSPEEQVQTLLNILSIFKTCRSSGCDLKSINGSAQAARIMISADLTGLSKKYSDIRLVEQSAS
  GLFVSKSQNLLEYL

  SEQ ID NO: 320

  MTKKEQPYNIGLDIGTSSVGWAVTNDNYDLLNIKKKNLWGVRLFEEAQTAKETRLNRSTRRRYR
  RRKNRINWLNEIFSEELAKTDPSFLIRLQNSWVSKKDPDRKRDKYNLFIDGPYTDKEYYREFPT
  IFHLRKELILNKDKADIRLIYLALHNILKYRGNFTYEHQKFNISNLNNNLSKELIELNQQLIKY
  DISFPDDCDWNHISDILIGRGNATQKSSNILKDFTLDKETKKLLKEVINLILGNVAHLNTIFKT
  SLTKDEEKLNFSGKDIESKLDDLDSILDDDQFTVLDAANRIYSTITLNEILNGESYFSMAKVNQ
  YENHAIDLCKLRDMWHTTKNEEAVEQSRQAYDDYINKPKYGTKELYTSLKKFLKVALPTNLAKE
  AEEKISKGTYLVKPRNSENGVVPYQLNKIEMEKIIDNQSQYYPFLKENKEKLLSILSFRIPYYV
  GPLQSAEKNPFAWMERKSNGHARPWNFDEIVDREKSSNKFIRRMTVTDSYLVGEPVLPKNSLIY
  QRYEVLNELNNIRITENLKTNPIGSRLTVETKQRIYNELFKKYKKVTVKKLTKWLIAQGYYKNP
  ILIGLSQKDEFNSTLTTYLDMKKIFGSSFMEDNKNYDQIEELIEWLTIFEDKQILNEKLHSSKY
  SYTPDQIKKISNMRYKGWGRLSKKILMDITTETNTPQLLQLSNYSILDLMWATNNNFISIMSND
  KYDFKNYIENHNLNKNEDQNISDLVNDIHVSPALKRGITQSIKIVQEIVKFMGHAPKHIFIEVT
  RETKKSEITTSREKRIKRLQSKLLNKANDFKPQLREYLVPNKKIQEELKKHKNDLSSERIMLYF
  LQNGKSLYSEESLNINKLSDYQVDHILPRTYIPDDSLENKALVLAKENQRKADDLLLNSNVIDR
  NLERWTYMLNNNMIGLKKFKNLTRRVITDKDKLGFIHRQLVQTSQMVKGVANILDNMYKNQGTT
  CIQARANLSTAFRKALSGQDDTYHFKHPELVKNRNVNDFHHAQDAYLASFLGTYRLRRFPTNEM
  LLMNGEYNKFYGQVKELYSKKKKLPDSRKNGFIISPLVNGTTQYDRNTGEIIWNVGFRDKILKI
  FNYHQCNVTRKTEIKTGQFYDQTIYSPKNPKYKKLIAQKKDMDPNIYGGFSGDNKSSITIVKID
  NNKIKPVAIPIRLINDLKDKKTLQNWLEENVKHKKSIQIIKNNVPIGQIIYSKKVGLLSLNSDR
  EVANRQQLILPPEHSALLRLLQIPDEDLDQILAFYDKNILVEILQELITKMKKFYPFYKGEREF
  LIANIENFNQATTSEKVNSLEELITLLHANSTSAHLIFNNIEKKAFGRKTHGLTLNNTDFIYQS
  VTGLYETRIHIE

  SEQ ID NO: 321

  MTKFNKNYSIGLDIGVSSVGYAVVTEDYRVPAFKFKVLGNTEKEKIKKNLIGSTTFVSAQPAKG
  TRVFRVNRRRIDRRNHRITYLRDIFQKEIEKVDKNFYRRLDESFRVLGDKSEDLQIKQPFFGDK
  ELETAYHKKYPTIYHLRKHLADADKNSPVADIREVYMAISHILKYRGHFLTLDKINPNNINMQN
  SWIDFIESCQEVFDLEISDESKNIADIFKSSENRQEKVKKILPYFQQELLKKDKSIFKQLLQLL
  FGLKTKFKDCFELEEEPDLNFSKENYDENLENFLGSLEEDFSDVFAKLKVLRDTILLSGMLTYT
  GATHARFSATMVERYEEHRKDLQRFKFFIKQNLSEQDYLDIFGRKTQNGFDVDKETKGYVGYIT
  NKMVLTNPQKQKTIQQNFYDYISGKITGIEGAEYFLNKISDGTFLRKLRTSDNGAIPNQIHAYE
  LEKIIERQGKDYPFLLENKDKLLSILTFKIPYYVGPLAKGSNSRFAWIKRATSSDILDDNDEDT
  RNGKIRPWNYQKLINMDETRDAFITNLIGNDIILLNEKVLPKRSLIYEEVMLQNELTRVKYKDK
  YGKAHFFDSELRQNIINGLFKNNSKRVNAKSLIKYLSDNHKDLNAIEIVSGVEKGKSFNSTLKT
  YNDLKTIFSEELLDSEIYQKELEEIIKVITVFDDKKSIKNYLTKFFGHLEILDEEKINQLSKLR
  YSGWGRYSAKLLLDIRDEDTGFNLLQFLRNDEENRNLTKLISDNTLSFEPKIKDIQSKSTIEDD
  IFDEIKKLAGSPAIKRGILNSIKIVDELVQIIGYPPHNIVIEMARENMTTEEGQKKAKTRKTKL
  ESALKNIENSLLENGKVPHSDEQLQSEKLYLYYLQNGKDMYTLDKTGSPAPLYLDQLDQYEVDH
  IIPYSFLPIDSIDNKVLTHRENNQQKLNNIPDKETVANMKPFWEKLYNAKLISQTKYQRLTTSE
  RTPDGVLTESMKAGFIERQLVETRQIIKHVARILDNRFSDTKIITLKSQLITNFRNTFHIAKIR
  ELNDYHHAHDAYLAVVVGQTLLKVYPKLAPELIYGHHAHFNRHEENKATLRKHLYSNIMRFFNN
  PDSKVSKDIWDCNRDLPIIKDVIYNSQINFVKRTMIKKGAFYNQNPVGKFNKQLAANNRYPLKT
  KALCLDTSIYGGYGPMNSALSIIIIAERFNEKKGKIETVKEFHDIFIIDYEKFNNNPFQFLNDT
  SENGFLKKNNINRVLGFYRIPKYSLMQKIDGTRMLFESKSNLHKATQFKLTKTQNELFFHMKRL
  LTKSNLMDLKSKSAIKESQNFILKHKEEFDNISNQLSAFSQKMLGNTTSLKNLIKGYNERKIKE
  IDIRDETIKYFYDNFIKMFSFVKSGAPKDINDFFDNKCTVARMRPKPDKKLLNATLIHQSITGL
  YETRIDLSKLGED

  SEQ ID NO: 322

  MKQEYFLGLDMGTGSLGWAVTDSTYQVMRKHGKALWGTRLFESASTAEERRMFRTARRRLDRRN
  WRIQVLQEIFSEEISKVDPGFFLRMKESKYYPEDKRDAEGNCPELPYALFVDDNYTDKNYHKDY
  PTIYHLRKMLMETTEIPDIRLVYLVLHHMMKHRGHFLLSGDISQIKEFKSTFEQLIQNIQDEEL
  EWHISLDDAAIQFVEHVLKDRNLTRSTKKSRLIKQLNAKSACEKAILNLLSGGTVKLSDIFNNK
  ELDESERPKVSFADSGYDDYIGIVEAELAEQYYIIASAKAVYDWSVLVEILGNSVSISEAKIKV
  YQKHQADLKTLKKIVRQYMTKEDYKRVFVDTEEKLNNYSAYIGMTKKNGKKVDLKSKQCTQADF
  YDFLKKNVIKVIDHKEITQEIESEIEKENFLPKQVTKDNGVIPYQVHDYELKKILDNLGTRMPF
  IKENAEKIQQLFEFRIPYYVGPLNRVDDGKDGKFTWSVRKSDARIYPWNFTEVIDVEASAEKFI
  RRMTNKCTYLVGEDVLPKDSLVYSKFMVLNELNNLRLNGEKISVELKQRIYEELFCKYRKVTRK
  KLERYLVIEGIAKKGVEITGIDGDFKASLTAYHDFKERLTDVQLSQRAKEAIVLNVVLFGDDKK
  LLKQRLSKMYPNLTTGQLKGICSLSYQGWGRLSKTFLEEITVPAPGTGEVWNIMTALWQTNDNL
  MQLLSRNYGFTNEVEEFNTLKKETDLSYKTVDELYVSPAVKRQIWQTLKVVKEIQKVMGNAPKR
  VFVEMAREKQEGKRSDSRKKQLVELYRACKNEERDWITELNAQSDQQLRSDKLFLYYIQKGRCM
  YSGETIQLDELWDNTKYDIDHIYPQSKTMDDSLNNRVLVKKNYNAIKSDTYPLSLDIQKKMMSF
  WKMLQQQGFITKEKYVRLVRSDELSADELAGFIERQIVETRQSTKAVATILKEALPDTEIVYVK
  AGNVSNFRQTYELLKVREMNDLHHAKDAYLNIVVGNAYFVKFTKNAAWFIRNNPGRSYNLKRMF
  EFDIERSGEIAWKAGNKGSIVTVKKVMQKNNILVTRKAYEVKGGLFDQQIMKKGKGQVPIKGND
  ERLADIEKYGGYNKAAGTYFMLVKSLDKKGKEIRTIEFVPLYLKNQIEINHESAIQYLAQERGL
  NSPEILLSKIKIDTLFKVDGFKMWLSGRTGNQLIFKGANQLILSHQEAAILKGVVKYVNRKNEN
  KDAKLSERDGMTEEKLLQLYDTFLDKLSNTVYSIRLSAQIKTLTEKRAKFIGLSNEDQCIVLNE
  ILHMFQCQSGSANLKLIGGPGSAGILVMNNNITACKQISVINQSPTGIYEKEIDLIKL

  SEQ ID NO: 323

  MKKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMKVLGNTDKSHIEKNLLGALLFDSGNTAEDRRL
  KRTARRRYTRRRNRILYLQEIFSEEMGKVDDSFFHRLEDSFLVTEDKRGERHPIFGNLEEEVKY
  HENFPTIYHLRQYLADNPEKVDLRLVYLALAHIIKFRGHFLIEGKFDTRNNDVQRLFQEFLAVY
  DNTFENSSLQEQNVQVEEILTDKISKSAKKDRVLKLFPNEKSNGRFAEFLKLIVGNQADFKKHF
  ELEEKAPLQFSKDTYEEELEVLLAQIGDNYAELFLSAKKLYDSILLSGILTVTDVGTKAPLSAS
  MIQRYNEHQMDLAQLKQFIRQKLSDKYNEVFSDVSKDGYAGYIDGKTNQEAFYKYLKGLLNKIE
  GSGYFLDKIEREDFLRKQRTFDNGSIPHQIHLQEMRAIIRRQAEFYPFLADNQDRIEKLLTFRI
  PYYVGPLARGKSDFAWLSRKSADKITPWNFDEIVDKESSAEAFINRMTNYDLYLPNQKVLPKHS
  LLYEKFTVYNELTKVKYKTEQGKTAFFDANMKQEIFDGVFKVYRKVTKDKLMDFLEKEFDEFRI
  VDLTGLDKENKVFNASYGTYHDLCKILDKDFLDNSKNEKILEDIVLTLTLFEDREMIRKRLENY
  SDLLTKEQVKKLERRHYTGWGRLSAELIHGIRNKESRKTILDYLIDDGNSNRNFMQLINDDALS
  FKEEIAKAQVIGETDNLNQVVSDIAGSPAIKKGILQSLKIVDELVKIMGHQPENIVVEMARENQ
  FTNQGRRNSQQRLKGLTDSIKEFGSQILKEHPVENSQLQNDRLFLYYLQNGRDMYTGEELDIDY
  LSQYDIDHIIPQAFIKDNSIDNRVLTSSKENRGKSDDVPSKDVVRKMKSYWSKLLSAKLITQRK
  FDNLTKAERGGLTDDDKAGFIKRQLVETRQITKHVARILDERFNTETDENNKKIRQVKIVTLKS
  NLVSNFRKEFELYKVREINDYHHAHDAYLNAVIGKALLGVYPQLEPEFVYGDYPHFHGHKENKA
  TAKKFFYSNIMNFFKKDDVRTDKNGEIIWKKDEHISNIKKVLSYPQVNIVKKVEEQTGGFSKES
  ILPKGNSDKLIPRKTKKFYWDTKKYGGFDSPIVAYSILVIADIEKGKSKKLKTVKALVGVTIME
  KMTFERDPVAFLERKGYRNVQEENIIKLPKYSLFKLENGRKRLLASARELQKGNEIVLPNHLGT
  LLYHAKNIHKVDEPKHLDYVDKHKDEFKELLDVVSNFSKKYTLAEGNLEKIKELYAQNNGEDLK
  ELASSFINLLTFTAIGAPATFKFFDKNIDRKRYTSTTEILNATLIHQSITGLYETRIDLNKLGG
  D

  SEQ ID NO: 324

  MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL
  KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY
  HEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY
  NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF
  DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS
  MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
  GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI
  PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS
  LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
  SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA
  HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF
  KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ
  TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR
  LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK
  FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS
  KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK
  SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS
  MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG
  KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLAS
  AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV
  ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
  ATLIHQSITGLYETRIDLSQLGGD

  SEQ ID NO: 325

  MTKPYSIGLDIGTNSVGWAVTTDNYKVPSKKMKVLGNTSKKYIKKNLLGVLLFDSGITAEGRRL
  KRTARRRYTRRRNRILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDSKYPIFGNLVEEKAY
  HDEFPTIYHLRKYLADSTKKADLRLVYLALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDFLDTY
  NAIFESDLSLENSKQLEEIVKDKISKLEKKDRILKLFPGEKNSGIFSEFLKLIVGNQADFRKCF
  NLDEKASLHFSKESYDEDLETLLGYIGDDYSDVFLKAKKLYDAILLSGFLTVTDNETEAPLSSA
  MIKRYNEHKEDLALLKEYIRNISLKTYNEVFKDDTKNGYAGYIDGKTNQEDFYVYLKKLLAEFE
  GADYFLEKIDREDFLRKQRTFDNGSIPYQIHLQEMRAILDKQAKFYPFLAKNKERIEKILTFRI
  PYYVGPLARGNSDFAWSIRKRNEKITPWNFEDVIDKESSAEAFINRMTSFDLYLPEEKVLPKHS
  LLYETFNVYNELTKVRFIAESMRDYQFLDSKQKKDIVRLYFKDKRKVTDKDIIEYLHAIYGYDG
  IELKGIEKQFNSSLSTYHDLLNIINDKEFLDDSSNEAIIEEIIHTLTIFEDREMIKQRLSKFEN
  IFDKSVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYLIDDGISNRNFMQLIHDDALSFK
  KKIQKAQIIGDEDKGNIKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGGRKPESIVVEMAREN
  QYTNQGKSNSQQRLKRLEKSLKELGSKILKENIPAKLSKIDNNALQNDRLYLYYLQNGKDMYTG
  DDLDIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGKSDDVPSLEVVKKRKTFWYQLLKS
  KLISQRKFDNLTKAERGGLSPEDKAGFIQRQLVETRQITKHVARLLDEKFNNKKDENNRAVRTV
  KIITLKSTLVSQFRKDFELYKVREINDFHHAHDAYLNAVVASALLKKYPKLEPEFVYGDYPKYN
  SFRERKSATEKVYFYSNIMNIFKKSISLADGRVIERPLIEVNEETGESVWNKESDLATVRRVLS
  YPQVNVVKKVEEQNHGLDRGKPKGLFNANLSSKPKPNSNENLVGAKEYLDPKKYGGYAGISNSF
  TVLVKGTIEKGAKKKITNVLEFQGISILDRINYRKDKLNFLLEKGYKDIELIIELPKYSLFELS
  DGSRRMLASILSTNNKRGEIHKGNQIFLSQKFVKLLYHAKRISNTINENHRKYVENHKKEFEEL
  FYYILEFNENYVGAKKNGKLLNSAFQSWQNHSIDELCSSFIGPTGSERKGLFELTSRGSAADFE
  FLGVKIPRYRDYTPSSLLKDATLIHQSVTGLYETRIDLAKLGEG

  SEQ ID NO: 326

  MKKQKFSDYYLGFDIGTNSVGWCVTDLDYNVLRFNKKDMWGSRLFDEAKTAAERRVQRNSRRRL
  KRRKWRLNLLEEIFSDEIMKIDSNFFRRLKESSLWLEDKNSKEKFTLFNDDNYKDYDFYKQYPT
  IFHLRDELIKNPEKKDIRLIYLALHSIFKSRGHFLFEGQNLKEIKNFETLYNNLISFLEDNGIN
  KSIDKDNIEKLEKIICDSGKGLKDKEKEFKGIFNSDKQLVAIFKLSVGSSVSLNDLFDTDEYKK
  EEVEKEKISFREQIYEDDKPIYYSILGEKIELLDIAKSFYDFMVLNNILSDSNYISEAKVKLYE
  EHKKDLKNLKYIIRKYNKENYDKLFKDKNENNYPAYIGLNKEKDKKEVVEKSRLKIDDLIKVIK
  GYLPKPERIEEKDKTIFNEILNKIELKTILPKQRISDNGTLPYQIHEVELEKILENQSKYYDFL
  NYEENGVSTKDKLLKTFKFRIPYYVGPLNSYHKDKGGNSWIVRKEEGKILPWNFEQKVDIEKSA
  EEFIKRMTNKCTYLNGEDVIPKDSFLYSEYIILNELNKVQVNDEFLNEENKRKIIDELFKENKK
  VSEKKFKEYLLVNQIANRTVELKGIKDSFNSNYVSYIKFKDIFGEKLNLDIYKEISEKSILWKC
  LYGDDKKIFEKKIKNEYGDILNKDEIKKINSFKFNTWGRLSEKLLTGIEFINLETGECYSSVME
  ALRRTNYNLMELLSSKFTLQESIDNENKEMNEVSYRDLIEESYVSPSLKRAILQTLKIYEEIKK
  ITGRVPKKVFIEMARGGDESMKNKKIPARQEQLKKLYDSCGNDIANFSIDIKEMKNSLSSYDNN
  SLRQKKLYLYYLQFGKCMYTGREIDLDRLLQNNDTYDIDHIYPRSKVIKDDSFDNLVLVLKNEN
  AEKSNEYPVKKEIQEKMKSFWRFLKEKNFISDEKYKRLTGKDDFELRGFMARQLVNVRQTTKEV
  GKILQQIEPEIKIVYSKAEIASSFREMFDFIKVRELNDTHHAKDAYLNIVAGNVYNTKFTEKPY
  RYLQEIKENYDVKKIYNYDIKNAWDKENSLEIVKKNMEKNTVNITRFIKEEKGELFNLNPIKKG
  ETSNEIISIKPKLYDGKDNKLNEKYGYYTSLKAAYFIYVEHEKKNKKVKTFERITRIDSTLIKN
  EKNLIKYLVSQKKLLNPKIIKKIYKEQTLIIDSYPYTFTGVDSNKKVELKNKKQLYLEKKYEQI
  LKNALKFVEDNQGETEENYKFIYLKKRNNNEKNETIDAVKERYNIEFNEMYDKFLEKLSSKDYK
  NYINNKLYTNFLNSKEKFKKLKLWEKSLILREFLKIFNKNTYGKYEIKDSQTKEKLFSFPEDTG
  RIRLGQSSLGNNKELLEESVTGLFVKKIKL

  SEQ ID NO: 327

  MKNYTIGLDIGVASVGWVCIDENYKILNYNNRHAFGVHEFESAESAAGRRLKRGMRRRYNRRKK
  RLQLLQSLFDSYITDSGFFSKTDSQHFWKNNNEFENRSLTEVLSSLRISSRKYPTIYHLRSDLI
  ESNKKMDLRLVYLALHNLVKYRGHFLQEGNWSEAASAEGMDDQLLELVTRYAELENLSPLDLSE
  SQWKAAETLLLNRNLTKTDQSKELTAMFGKEYEPFCKLVAGLGVSLHQLFPSSEQALAYKETKT
  KVQLSNENVEEVMELLLEEESALLEAVQPFYQQVVLYELLKGETYVAKAKVSAFKQYQKDMASL
  KNLLDKTFGEKVYRSYFISDKNSQREYQKSHKVEVLCKLDQFNKEAKFAETFYKDLKKLLEDKS
  KTSIGTTEKDEMLRIIKAIDSNQFLQKQKGIQNAAIPHQNSLYEAEKILRNQQAHYPFITTEWI
  EKVKQILAFRIPYYIGPLVKDTTQSPFSWVERKGDAPITPWNFDEQIDKAASAEAFISRMRKTC
  TYLKGQEVLPKSSLTYERFEVLNELNGIQLRTTGAESDFRHRLSYEMKCWIIDNVFKQYKTVST
  KRLLQELKKSPYADELYDEHTGEIKEVFGTQKENAFATSLSGYISMKSILGAVVDDNPAMTEEL
  IYWIAVFEDREILHLKIQEKYPSITDVQRQKLALVKLPGWGRFSRLLIDGLPLDEQGQSVLDHM
  EQYSSVFMEVLKNKGFGLEKKIQKMNQHQVDGTKKIRYEDIEELAGSPALKRGIWRSVKIVEEL
  VSIFGEPANIVLEVAREDGEKKRTKSRKDQWEELTKTTLKNDPDLKSFIGEIKSQGDQRFNEQR
  FWLYVTQQGKCLYTGKALDIQNLSMYEVDHILPQNFVKDDSLDNLALVMPEANQRKNQVGQNKM
  PLEIIEANQQYAMRTLWERLHELKLISSGKLGRLKKPSFDEVDKDKFIARQLVETRQIIKHVRD
  LLDERFSKSDIHLVKAGIVSKFRRFSEIPKIRDYNNKHHAMDALFAAALIQSILGKYGKNFLAF
  DLSKKDRQKQWRSVKGSNKEFFLFKNFGNLRLQSPVTGEEVSGVEYMKHVYFELPWQTTKMTQT
  GDGMFYKESIFSPKVKQAKYVSPKTEKFVHDEVKNHSICLVEFTFMKKEKEVQETKFIDLKVIE
  HHQFLKEPESQLAKFLAEKETNSPIIHARIIRTIPKYQKIWIEHFPYYFISTRELHNARQFEIS
  YELMEKVKQLSERSSVEELKIVFGLLIDQMNDNYPIYTKSSIQDRVQKFVDTQLYDFKSFEIGF
  EELKKAVAANAQRSDTFGSRISKKPKPEEVAIGYESITGLKYRKPRSVVGTKR

  SEQ ID NO: 328

  MKKEIKDYFLGLDVGTGSVGWAVTDTDYKLLKANRKDLWGMRCFETAETAEVRRLHRGARRRIE
  RRKKRIKLLQELFSQEIAKTDEGFFQRMKESPFYAEDKTILQENTLFNDKDFADKTYHKAYPTI
  NHLIKAWIENKVKPDPRLLYLACHNIIKKRGHFLFEGDFDSENQFDTSIQALFEYLREDMEVDI
  DADSQKVKEILKDSSLKNSEKQSRLNKILGLKPSDKQKKAITNLISGNKINFADLYDNPDLKDA
  EKNSISFSKDDFDALSDDLASILGDSFELLLKAKAVYNCSVLSKVIGDEQYLSFAKVKIYEKHK
  TDLTKLKNVIKKHFPKDYKKVFGYNKNEKNNNNYSGYVGVCKTKSKKLIINNSVNQEDFYKFLK
  TILSAKSEIKEVNDILTEIETGTFLPKQISKSNAEIPYQLRKMELEKILSNAEKHFSFLKQKDE
  KGLSHSEKIIMLLTFKIPYYIGPINDNHKKFFPDRCWVVKKEKSPSGKTTPWNFFDHIDKEKTA
  EAFITSRTNFCTYLVGESVLPKSSLLYSEYTVLNEINNLQIIIDGKNICDIKLKQKIYEDLFKK
  YKKITQKQISTFIKHEGICNKTDEVIILGIDKECTSSLKSYIELKNIFGKQVDEISTKNMLEEI
  IRWATIYDEGEGKTILKTKIKAEYGKYCSDEQIKKILNLKFSGWGRLSRKFLETVTSEMPGFSE
  PVNIITAMRETQNNLMELLSSEFTFTENIKKINSGFEDAEKQFSYDGLVKPLFLSPSVKKMLWQ
  TLKLVKEISHITQAPPKKIFIEMAKGAELEPARTKTRLKILQDLYNNCKNDADAFSSEIKDLSG
  KIENEDNLRLRSDKLYLYYTQLGKCMYCGKPIEIGHVFDTSNYDIDHIYPQSKIKDDSISNRVL
  VCSSCNKNKEDKYPLKSEIQSKQRGFWNFLQRNNFISLEKLNRLTRATPISDDETAKFIARQLV
  ETRQATKVAAKVLEKMFPETKIVYSKAETVSMFRNKFDIVKCREINDFHHAHDAYLNIVVGNVY
  NTKFTNNPWNFIKEKRDNPKIADTYNYYKVFDYDVKRNNITAWEKGKTIITVKDMLKRNTPIYT
  RQAACKKGELFNQTIMKKGLGQHPLKKEGPFSNISKYGGYNKVSAAYYTLIEYEEKGNKIRSLE
  TIPLYLVKDIQKDQDVLKSYLTDLLGKKEFKILVPKIKINSLLKINGFPCHITGKTNDSFLLRP
  AVQFCCSNNEVLYFKKIIRFSEIRSQREKIGKTISPYEDLSFRSYIKENLWKKTKNDEIGEKEF
  YDLLQKKNLEIYDMLLTKHKDTIYKKRPNSATIDILVKGKEKFKSLIIENQFEVILEILKLFSA
  TRNVSDLQHIGGSKYSGVAKIGNKISSLDNCILIYQSITGIFEKRIDLLKV

  SEQ ID NO: 329

  MEGQMKNNGNNLQQGNYYLGLDVGTSSVGWAVTDTDYNVLKFRGKSMWGARLFDEASTAEERRT
  HRGNRRRLARRKYRLLLLEQLFEKEIRKIDDNFFVRLHESNLWADDKSKPSKFLLFNDTNFTDK
  DYLKKYPTIYHLRSDLIHNSTEHDIRLVFLALHHLIKYRGHFIYDNSANGDVKTLDEAVSDFEE
  YLNENDIEFNIENKKEFINVLSDKHLTKKEKKISLKKLYGDITDSENINISVLIEMLSGSSISL
  SNLFKDIEFDGKQNLSLDSDIEETLNDVVDILGDNIDLLIHAKEVYDIAVLTSSLGKHKYLCDA
  KVELFEKNKKDLMILKKYIKKNHPEDYKKIFSSPTEKKNYAAYSQTNSKNVCSQEEFCLFIKPY
  IRDMVKSENEDEVRIAKEVEDKSFLTKLKGTNNSVVPYQIHERELNQILKNIVAYLPFMNDEQE
  DISVVDKIKLIFKFKIPYYVGPLNTKSTRSWVYRSDEKIYPWNFSNVIDLDKTAHEFMNRLIGR
  CTYTNDPVLPMDSLLYSKYNVLNEINPIKVNGKAIPVEVKQAIYTDLFENSKKKVTRKSIYIYL
  LKNGYIEKEDIVSGIDIEIKSKLKSHHDFTQIVQENKCTPEEIERIIKGILVYSDDKSMLRRWL
  KNNIKGLSENDVKYLAKLNYKEWGRLSKTLLTDIYTINPEDGEACSILDIMWNTNATLMEILSN
  EKYQFKQNIENYKAENYDEKQNLHEELDDMYISPAARRSIWQALRIVDEIVDIKKSAPKKIFIE
  MAREKKSAMKKKRTESRKDTLLELYKSCKSQADGFYDEELFEKLSNESNSRLRRDQLYLYYTQM
  GRSMYTGKRIDFDKLINDKNTYDIDHIYPRSKIKDDSITNRVLVEKDINGEKTDIYPISEDIRQ
  KMQPFWKILKEKGLINEEKYKRLTRNYELTDEELSSFVARQLVETQQSTKALATLLKKEYPSAK
  IVYSKAGNVSEFRNRKDKELPKFREINDLHHAKDAYLNIVVGNVYDTKFTEKFFNNIRNENYSL
  KRVFDFSVPGAWDAKGSTFNTIKKYMAKNNPIIAFAPYEVKGELFDQQIVPKGKGQFPIKQGKD
  IEKYGGYNKLSSAFLFAVEYKGKKARERSLETVYIKDVELYLQDPIKYCESVLGLKEPQIIKPK
  ILMGSLFSINNKKLVVTGRSGKQYVCHHIYQLSINDEDSQYLKNIAKYLQEEPDGNIERQNILN
  ITSVNNIKLFDVLCTKFNSNTYEIILNSLKNDVNEGREKFSELDILEQCNILLQLLKAFKCNRE
  SSNLEKLNNKKQAGVIVIPHLFTKCSVFKVIHQSITGLFEKEMDLLK

  SEQ ID NO: 330

  MGRKPYILSLDIGTGSVGYACMDKGFNVLKYHDKDALGVYLFDGALTAQERRQFRTSRRRKNRR
  IKRLGLLQELLAPLVQNPNFYQFQRQFAWKNDNMDFKNKSLSEVLSFLGYESKKYPTIYHLQEA
  LLLKDEKFDPELIYMALYHLVKYRGHFLFDHLKIENLTNNDNMHDFVELIETYENLNNIKLNLD
  YEKTKVIYEILKDNEMTKNDRAKRVKNMEKKLEQFSIMLLGLKFNEGKLFNHADNAEELKGANQ
  SHTFADNYEENLTPFLTVEQSEFIERANKIYLSLTLQDILKGKKSMAMSKVAAYDKFRNELKQV
  KDIVYKADSTRTQFKKIFVSSKKSLKQYDATPNDQTFSSLCLFDQYLIRPKKQYSLLIKELKKI
  IPQDSELYFEAENDTLLKVLNTTDNASIPMQINLYEAETILRNQQKYHAEITDEMIEKVLSLIQ
  FRIPYYVGPLVNDHTASKFGWMERKSNESIKPWNFDEVVDRSKSATQFIRRMTNKCSYLINEDV
  LPKNSLLYQEMEVLNELNATQIRLQTDPKNRKYRMMPQIKLFAVEHIFKKYKTVSHSKFLEIML
  NSNHRENFMNHGEKLSIFGTQDDKKFASKLSSYQDMTKIFGDIEGKRAQIEEIIQWITIFEDKK
  ILVQKLKECYPELTSKQINQLKKLNYSGWGRLSEKLLTHAYQGHSIIELLRHSDENFMEILTND
  VYGFQNFIKEENQVQSNKIQHQDIANLTTSPALKKGIWSTIKLVRELTSIFGEPEKIIMEFATE
  DQQKGKKQKSRKQLWDDNIKKNKLKSVDEYKYIIDVANKLNNEQLQQEKLWLYLSQNGKCMYSG
  QSIDLDALLSPNATKHYEVDHIFPRSFIKDDSIDNKVLVIKKMNQTKGDQVPLQFIQQPYERIA
  YWKSLNKAGLISDSKLHKLMKPEFTAMDKEGFIQRQLVETRQISVHVRDFLKEEYPNTKVIPMK
  AKMVSEFRKKFDIPKIRQMNDAHHAIDAYLNGVVYHGAQLAYPNVDLFDFNFKWEKVREKWKAL
  GEFNTKQKSRELFFFKKLEKMEVSQGERLISKIKLDMNHFKINYSRKLANIPQQFYNQTAVSPK
  TAELKYESNKSNEVVYKGLTPYQTYVVAIKSVNKKGKEKMEYQMIDHYVFDFYKFQNGNEKELA
  LYLAQRENKDEVLDAQIVYSLNKGDLLYINNHPCYFVSRKEVINAKQFELTVEQQLSLYNVMNN
  KETNVEKLLIEYDFIAEKVINEYHHYLNSKLKEKRVRTFFSESNQTHEDFIKALDELFKVVTAS
  ATRSDKIGSRKNSMTHRAFLGKGKDVKIAYTSISGLKTTKPKSLFKLAESRNEL

  SEQ ID NO: 331

  MAKILGLDLGTNSIGWAVVERENIDFSLIDKGVRIFSEGVKSEKGIESSRAAERTGYRSARKIK
  YRRKLRKYETLKVLSLNRMCPLSIEEVEEWKKSGFKDYPLNPEFLKWLSTDEESNVNPYFFRDR
  ASKHKVSLFELGRAFYHIAQRRGFLSNRLDQSAEGILEEHCPKIEAIVEDLISIDEISTNITDY
  FFETGILDSNEKNGYAKDLDEGDKKLVSLYKSLLAILKKNESDFENCKSEIIERLNKKDVLGKV
  KGKIKDISQAMLDGNYKTLGQYFYSLYSKEKIRNQYTSREEHYLSEFITICKVQGIDQINEEEK
  INEKKFDGLAKDLYKAIFFQRPLKSQKGLIGKCSFEKSKSRCAISHPDFEEYRMWTYLNTIKIG
  TQSDKKLRFLTQDEKLKLVPKFYRKNDFNFDVLAKELIEKGSSFGFYKSSKKNDFFYWFNYKPT
  DTVAACQVAASLKNAIGEDWKTKSFKYQTINSNKEQVSRTVDYKDLWHLLTVATSDVYLYEFAI
  DKLGLDEKNAKAFSKTKLKKDFASLSLSAINKILPYLKEGLLYSHAVFVANIENIVDENIWKDE
  KQRDYIKTQISEIIENYTLEKSRFEIINGLLKEYKSENEDGKRVYYSKEAEQSFENDLKKKLVL
  FYKSNEIENKEQQETIFNELLPIFIQQLKDYEFIKIQRLDQKVLIFLKGKNETGQIFCTEEKGT
  AEEKEKKIKNRLKKLYHPSDIEKFKKKIIKDEFGNEKIVLGSPLTPSIKNPMAMRALHQLRKVL
  NALILEGQIDEKTIIHIEMARELNDANKRKGIQDYQNDNKKFREDAIKEIKKLYFEDCKKEVEP
  TEDDILRYQLWMEQNRSEIYEEGKNISICDIIGSNPAYDIEHTIPRSRSQDNSQMNKTLCSQRF
  NREVKKQSMPIELNNHLEILPRIAHWKEEADNLTREIEIISRSIKAAATKEIKDKKIRRRHYLT
  LKRDYLQGKYDRFIWEEPKVGFKNSQIPDTGIITKYAQAYLKSYFKKVESVKGGMVAEFRKIWG
  IQESFIDENGMKHYKVKDRSKHTHHTIDAITIACMTKEKYDVLAHAWTLEDQQNKKEARSIIEA
  SKPWKTFKEDLLKIEEEILVSHYTPDNVKKQAKKIVRVRGKKQFVAEVERDVNGKAVPKKAASG
  KTIYKLDGEGKKLPRLQQGDTIRGSLHQDSIYGAIKNPLNTDEIKYVIRKDLESIKGSDVESIV
  DEVVKEKIKEAIANKVLLLSSNAQQKNKLVGTVWMNEEKRIAINKVRIYANSVKNPLHIKEHSL
  LSKSKHVHKQKVYGQNDENYAMAIYELDGKRDFELINIFNLAKLIKQGQGFYPLHKKKEIKGKI
  VFVPIEKRNKRDVVLKRGQQVVFYDKEVENPKDISEIVDFKGRIYIIEGLSIQRIVRPSGKVDE
  YGVIMLRYFKEARKADDIKQDNFKPDGVFKLGENKPTRKMNHQFTAFVEGIDFKVLPSGKFEKI

  SEQ ID NO: 332

  MEFKKVLGLDIGTNSIGCALLSLPKSIQDYGKGGRLEWLTSRVIPLDADYMKAFIDGKNGLPQV
  ITPAGKRRQKRGSRRLKHRYKLRRSRLIRVFKTLNWLPEDFPLDNPKRIKETISTEGKFSFRIS
  DYVPISDESYREFYREFGYPENEIEQVIEEINFRRKTKGKNKNPMIKLLPEDWVVYYLRKKALI
  KPTTKEELIRIIYLFNQRRGFKSSRKDLTETAILDYDEFAKRLAEKEKYSAENYETKFVSITKV
  KEVVELKTDGRKGKKRFKVILEDSRIEPYEIERKEKPDWEGKEYTFLVTQKLEKGKFKQNKPDL
  PKEEDWALCTTALDNRMGSKHPGEFFFDELLKAFKEKRGYKIRQYPVNRWRYKKELEFIWTKQC
  QLNPELNNLNINKEILRKLATVLYPSQSKFFGPKIKEFENSDVLHIISEDIIYYQRDLKSQKSL
  ISECRYEKRKGIDGEIYGLKCIPKSSPLYQEFRIWQDIHNIKVIRKESEVNGKKKINIDETQLY
  INENIKEKLFELFNSKDSLSEKDILELISLNIINSGIKISKKEEETTHRINLFANRKELKGNET
  KSRYRKVFKKLGFDGEYILNHPSKLNRLWHSDYSNDYADKEKTEKSILSSLGWKNRNGKWEKSK
  NYDVFNLPLEVAKAIANLPPLKKEYGSYSALAIRKMLVVMRDGKYWQHPDQIAKDQENTSLMLF
  DKNLIQLTNNQRKVLNKYLLTLAEVQKRSTLIKQKLNEIEHNPYKLELVSDQDLEKQVLKSFLE
  KKNESDYLKGLKTYQAGYLIYGKHSEKDVPIVNSPDELGEYIRKKLPNNSLRNPIVEQVIRETI
  FIVRDVWKSFGIIDEIHIELGRELKNNSEERKKTSESQEKNFQEKERARKLLKELLNSSNFEHY
  DENGNKIFSSFTVNPNPDSPLDIEKFRIWKNQSGLTDEELNKKLKDEKIPTEIEVKKYILWLTQ
  KCRSPYTGKIIPLSKLFDSNVYEIEHIIPRSKMKNDSTNNLVICELGVNKAKGDRLAANFISES
  NGKCKFGEVEYTLLKYGDYLQYCKDTFKYQKAKYKNLLATEPPEDFIERQINDTRYIGRKLAEL
  LTPVVKDSKNIIFTIGSITSELKITWGLNGVWKDILRPRFKRLESIINKKLIFQDEDDPNKYHF
  DLSINPQLDKEGLKRLDHRHHALDATIIAATTREHVRYLNSLNAADNDEEKREYFLSLCNHKIR
  DFKLPWENFTSEVKSKLLSCVVSYKESKPILSDPFNKYLKWEYKNGKWQKVFAIQIKNDRWKAV
  RRSMFKEPIGTVWIKKIKEVSLKEAIKIQAIWEEVKNDPVRKKKEKYIYDDYAQKVIAKIVQEL
  GLSSSMRKQDDEKLNKFINEAKVSAGVNKNLNTTNKTIYNLEGRFYEKIKVAEYVLYKAKRMPL
  NKKEYIEKLSLQKMFNDLPNFILEKSILDNYPEILKELESDNKYIIEPHKKNNPVNRLLLEHIL
  EYHNNPKEAFSTEGLEKLNKKAINKIGKPIKYITRLDGDINEEEIFRGAVFETDKGSNVYFVMY
  ENNQTKDREFLKPNPSISVLKAIEHKNKIDFFAPNRLGFSRIILSPGDLVYVPTNDQYVLIKDN
  SSNETIINWDDNEFISNRIYQVKKFTGNSCYFLKNDIASLILSYSASNGVGEFGSQNISEYSVD
  DPPIRIKDVCIKIRVDRLGNVRPL

  SEQ ID NO: 333

  MKHILGLDLGTNSIGWALIERNIEEKYGKIIGMGSRIVPMGAELSKFEQGQAQTKNADRRTNRG
  ARRLNKRYKQRRNKLIYILQKLDMLPSQIKLKEDFSDPNKIDKITILPISKKQEQLTAFDLVSL
  RVKALTEKVGLEDLGKIIYKYNQLRGYAGGSLEPEKEDIFDEEQSKDKKNKSFIAFSKIVFLGE
  PQEEIFKNKKLNRRAIIVETEEGNFEGSTFLENIKVGDSLELLINISASKSGDTITIKLPNKTN
  WRKKMENIENQLKEKSKEMGREFYISEFLLELLKENRWAKIRNNTILRARYESEFEAIWNEQVK
  HYPFLENLDKKTLIEIVSFIFPGEKESQKKYRELGLEKGLKYIIKNQVVFYQRELKDQSHLISD
  CRYEPNEKAIAKSHPVFQEYKVWEQINKLIVNTKIEAGTNRKGEKKYKYIDRPIPTALKEWIFE
  ELQNKKEITFSAIFKKLKAEFDLREGIDFLNGMSPKDKLKGNETKLQLQKSLGELWDVLGLDSI
  NRQIELWNILYNEKGNEYDLTSDRTSKVLEFINKYGNNIVDDNAEETAIRISKIKFARAYSSLS
  LKAVERILPLVRAGKYFNNDFSQQLQSKILKLLNENVEDPFAKAAQTYLDNNQSVLSEGGVGNS
  IATILVYDKHTAKEYSHDELYKSYKEINLLKQGDLRNPLVEQIINEALVLIRDIWKNYGIKPNE
  IRVELARDLKNSAKERATIHKRNKDNQTINNKIKETLVKNKKELSLANIEKVKLWEAQRHLSPY
  TGQPIPLSDLFDKEKYDVDHIIPISRYFDDSFTNKVISEKSVNQEKANRTAMEYFEVGSLKYSI
  FTKEQFIAHVNEYFSGVKRKNLLATSIPEDPVQRQIKDTQYIAIRVKEELNKIVGNENVKTTTG
  SITDYLRNHWGLTDKFKLLLKERYEALLESEKFLEAEYDNYKKDFDSRKKEYEEKEVLFEEQEL
  TREEFIKEYKENYIRYKKNKLIIKGWSKRIDHRHHAIDALIVACTEPAHIKRLNDLNKVLQDWL
  VEHKSEFMPNFEGSNSELLEEILSLPENERTEIFTQIEKFRAIEMPWKGFPEQVEQKLKEIIIS
  HKPKDKLLLQYNKAGDRQIKLRGQLHEGTLYGISQGKEAYRIPLTKFGGSKFATEKNIQKIVSP
  FLSGFIANHLKEYNNKKEEAFSAEGIMDLNNKLAQYRNEKGELKPHTPISTVKIYYKDPSKNKK
  KKDEEDLSLQKLDREKAFNEKLYVKTGDNYLFAVLEGEIKTKKTSQIKRLYDIISFFDATNFLK
  EEFRNAPDKKTFDKDLLFRQYFEERNKAKLLFTLKQGDFVYLPNENEEVILDKESPLYNQYWGD
  LKERGKNIYVVQKFSKKQIYFIKHTIADIIKKDVEFGSQNCYETVEGRSIKENCFKLEIDRLGN
  IVKVIKR

  SEQ ID NO: 334

  MHVEIDFPHFSRGDSHLAMNKNEILRGSSVLYRLGLDLGSNSLGWFVTHLEKRGDRHEPVALGP
  GGVRIFPDGRDPQSGTSNAVDRRMARGARKRRDRFVERRKELIAALIKYNLLPDDARERRALEV
  LDPYALRKTALTDTLPAHHVGRALFHLNQRRGFQSNRKTDSKQSEDGAIKQAASRLATDKGNET
  LGVFFADMHLRKSYEDRQTAIRAELVRLGKDHLTGNARKKIWAKVRKRLFGDEVLPRADAPHGV
  RARATITGTKASYDYYPTRDMLRDEFNAIWAGQSAHHATITDEARTEIEHIIFYQRPLKPAIVG
  KCTLDPATRPFKEDPEGYRAPWSHPLAQRFRILSEARNLEIRDTGKGSRRLTKEQSDLVVAALL
  ANREVKFDKLRTLLKLPAEARFNLESDRRAALDGDQTAARLSDKKGFNKAWRGFPPERQIAIVA
  RLEETEDENELIAWLEKECALDGAAAARVANTTLPDGHCRLGLRAIKKIVPIMQDGLDEDGVAG
  AGYHIAAKRAGYDHAKLPTGEQLGRLPYYGQWLQDAVVGSGDARDQKEKQYGQFPNPTVHIGLG
  QLRRVVNDLIDKYGPPTEISIEFTRALKLSEQQKAERQREQRRNQDKNKARAEELAKFGRPANP
  RNLLKMRLWEELAHDPLDRKCVYTGEQISIERLLSDEVDIDHILPVAMTLDDSPANKIICMRYA
  NRHKRKQTPSEAFGSSPTLQGHRYNWDDIAARATGLPRNKRWRFDANAREEFDKRGGFLARQLN
  ETGWLARLAKQYLGAVTDPNQIWVVPGRLTSMLRGKWGLNGLLPSDNYAGVQDKAEEFLASTDD
  MEFSGVKNRADHRHHAIDGLVTALTDRSLLWKMANAYDEEHEKFVIEPPWPTMRDDLKAALEKM
  VVSHKPDHGIEGKLHEDSAYGFVKPLDATGLKEEEAGNLVYRKAIESLNENEVDRIRDIQLRTI
  VRDHVNVEKTKGVALADALRQLQAPSDDYPQFKHGLRHVRILKKEKGDYLVPIANRASGVAYKA
  YSAGENFCVEVFETAGGKWDGEAVRRFDANKKNAGPKIAHAPQWRDANEGAKLVMRIHKGDLIR
  LDHEGRARIMVVHRLDAAAGRFKLADHNETGNLDKRHATNNDIDPFRWLMASYNTLKKLAAVPV
  RVDELGRVWRVMPN

  SEQ ID NO: 335

  METTLGIDLGTNSIGLALVDQEEHQILYSGVRIFPEGINKDTIGLGEKEESRNATRRAKRQMRR
  QYFRKKLRKAKLLELLIAYDMCPLKPEDVRRWKNWDKQQKSTVRQFPDTPAFREWLKQNPYELR
  KQAVTEDVTRPELGRILYQMIQRRGFLSSRKGKEEGKIFTGKDRMVGIDETRKNLQKQTLGAYL
  YDIAPKNGEKYRFRTERVRARYTLRDMYIREFEIIWQRQAGHLGLAHEQATRKKNIFLEGSATN
  VRNSKLITHLQAKYGRGHVLIEDTRITVTFQLPLKEVLGGKIEIEEEQLKFKSNESVLFWQRPL
  RSQKSLLSKCVFEGRNFYDPVHQKWIIAGPTPAPLSHPEFEEFRAYQFINNIIYGKNEHLTAIQ
  REAVFELMCTESKDFNFEKIPKHLKLFEKFNFDDTTKVPACTTISQLRKLFPHPVWEEKREEIW
  HCFYFYDDNTLLFEKLQKDYALQTNDLEKIKKIRLSESYGNVSLKAIRRINPYLKKGYAYSTAV
  LLGGIRNSFGKRFEYFKEYEPEIEKAVCRILKEKNAEGEVIRKIKDYLVHNRFGFAKNDRAFQK
  LYHHSQAITTQAQKERLPETGNLRNPIVQQGLNELRRTVNKLLATCREKYGPSFKFDHIHVEMG
  RELRSSKTEREKQSRQIRENEKKNEAAKVKLAEYGLKAYRDNIQKYLLYKEIEEKGGTVCCPYT
  GKTLNISHTLGSDNSVQIEHIIPYSISLDDSLANKTLCDATFNREKGELTPYDFYQKDPSPEKW
  GASSWEEIEDRAFRLLPYAKAQRFIRRKPQESNEFISRQLNDTRYISKKAVEYLSAICSDVKAF
  PGQLTAELRHLWGLNNILQSAPDITFPLPVSATENHREYYVITNEQNEVIRLFPKQGETPRTEK
  GELLLTGEVERKVFRCKGMQEFQTDVSDGKYWRRIKLSSSVTWSPLFAPKPISADGQIVLKGRI
  EKGVFVCNQLKQKLKTGLPDGSYWISLPVISQTFKEGESVNNSKLTSQQVQLFGRVREGIFRCH
  NYQCPASGADGNFWCTLDTDTAQPAFTPIKNAPPGVGGGQIILTGDVDDKGIFHADDDLHYELP
  ASLPKGKYYGIFTVESCDPTLIPIELSAPKTSKGENLIEGNIWVDEHTGEVRFDPKKNREDQRH
  HAIDAIVIALSSQSLFQRLSTYNARRENKKRGLDSTEHFPSPWPGFAQDVRQSVVPLLVSYKQN
  PKTLCKISKTLYKDGKKIHSCGNAVRGQLHKETVYGQRTAPGATEKSYHIRKDIRELKTSKHIG
  KVVDITIRQMLLKHLQENYHIDITQEFNIPSNAFFKEGVYRIFLPNKHGEPVPIKKIRMKEELG
  NAERLKDNINQYVNPRNNHHVMIYQDADGNLKEEIVSFWSVIERQNQGQPIYQLPREGRNIVSI
  LQINDTFLIGLKEEEPEVYRNDLSTLSKHLYRVQKLSGMYYTFRHHLASTLNNEREEFRIQSLE
  AWKRANPVKVQIDEIGRITFLNGPLC

  SEQ ID NO: 336

  MESSQILSPIGIDLGGKFTGVCLSHLEAFAELPNHANTKYSVILIDHNNFQLSQAQRRATRHRV
  RNKKRNQFVKRVALQLFQHILSRDLNAKEETALCHYLNNRGYTYVDTDLDEYIKDETTINLLKE
  LLPSESEHNFIDWFLQKMQSSEFRKILVSKVEEKKDDKELKNAVKNIKNFITGFEKNSVEGHRH
  RKVYFENIKSDITKDNQLDSIKKKIPSVCLSNLLGHLSNLQWKNLHRYLAKNPKQFDEQTFGNE
  FLRMLKNFRHLKGSQESLAVRNLIQQLEQSQDYISILEKTPPEITIPPYEARTNTGMEKDQSLL
  LNPEKLNNLYPNWRNLIPGIIDAHPFLEKDLEHTKLRDRKRIISPSKQDEKRDSYILQRYLDLN
  KKIDKFKIKKQLSFLGQGKQLPANLIETQKEMETHFNSSLVSVLIQIASAYNKEREDAAQGIWF
  DNAFSLCELSNINPPRKQKILPLLVGAILSEDFINNKDKWAKFKIFWNTHKIGRTSLKSKCKEI
  EEARKNSGNAFKIDYEEALNHPEHSNNKALIKIIQTIPDIIQAIQSHLGHNDSQALIYHNPFSL
  SQLYTILETKRDGFHKNCVAVTCENYWRSQKTEIDPEISYASRLPADSVRPFDGVLARMMQRLA
  YEIAMAKWEQIKHIPDNSSLLIPIYLEQNRFEFEESFKKIKGSSSDKTLEQAIEKQNIQWEEKF
  QRIINASMNICPYKGASIGGQGEIDHIYPRSLSKKHFGVIFNSEVNLIYCSSQGNREKKEEHYL
  LEHLSPLYLKHQFGTDNVSDIKNFISQNVANIKKYISFHLLTPEQQKAARHALFLDYDDEAFKT
  ITKFLMSQQKARVNGTQKFLGKQIMEFLSTLADSKQLQLEFSIKQITAEEVHDHRELLSKQEPK
  LVKSRQQSFPSHAIDATLTMSIGLKEFPQFSQELDNSWFINHLMPDEVHLNPVRSKEKYNKPNI
  SSTPLFKDSLYAERFIPVWVKGETFAIGFSEKDLFEIKPSNKEKLFTLLKTYSTKNPGESLQEL
  QAKSKAKWLYFPINKTLALEFLHHYFHKEIVTPDDTTVCHFINSLRYYTKKESITVKILKEPMP
  VLSVKFESSKKNVLGSFKHTIALPATKDWERLFNHPNFLALKANPAPNPKEFNEFIRKYFLSDN
  NPNSDIPNNGHNIKPQKHKAVRKVFSLPVIPGNAGTMMRIRRKDNKGQPLYQLQTIDDTPSMGI
  QINEDRLVKQEVLMDAYKTRNLSTIDGINNSEGQAYATFDNWLTLPVSTFKPEIIKLEMKPHSK
  TRRYIRITQSLADFIKTIDEALMIKPSDSIDDPLNMPNEIVCKNKLFGNELKPRDGKMKIVSTG
  KIVTYEFESDSTPQWIQTLYVTQLKKQP

  SEQ ID NO: 337

  MKKIVGLDLGTNSIGWALINAYINKEHLYGIEACGSRIIPMDAAILGNFDKGNSISQTADRTSY
  RGIRRLRERHLLRRERLHRILDLLGFLPKHYSDSLNRYGKFLNDIECKLPWVKDETGSYKFIFQ
  ESFKEMLANFTEHHPILIANNKKVPYDWTIYYLRKKALTQKISKEELAWILLNFNQKRGYYQLR
  GEEEETPNKLVEYYSLKVEKVEDSGERKGKDTWYNVHLENGMIYRRTSNIPLDWEGKTKEFIVT
  TDLEADGSPKKDKEGNIKRSFRAPKDDDWTLIKKKTEADIDKIKMTVGAYIYDTLLQKPDQKIR
  GKLVRTIERKYYKNELYQILKTQSEFHEELRDKQLYIACLNELYPNNEPRRNSISTRDFCHLFI
  EDIIFYQRPLKSKKSLIDNCPYEENRYIDKESGEIKHASIKCIAKSHPLYQEFRLWQFIVNLRI
  YRKETDVDVTQELLPTEADYVTLFEWLNEKKEIDQKAFFKYPPFGFKKTTSNYRWNYVEDKPYP
  CNETHAQIIARLGKAHIPKAFLSKEKEETLWHILYSIEDKQEIEKALHSFANKNNLSEEFIEQF
  KNFPPFKKEYGSYSAKAIKKLLPLMRMGKYWSIENIDNGTRIRINKIIDGEYDENIRERVRQKA
  INLTDITHFRALPLWLACYLVYDRHSEVKDIVKWKTPKDIDLYLKSFKQHSLRNPIVEQVITET
  LRTVRDIWQQVGHIDEIHIELGREMKNPADKRARMSQQMIKNENTNLRIKALLTEFLNPEFGIE
  NVRPYSPSQQDLLRIYEEGVLNSILELPEDIGIILGKFNQTDTLKRPTRSEILRYKLWLEQKYR
  SPYTGEMIPLSKLFTPAYEIEHIIPQSRYFDDSLSNKVICESEINKLKDRSLGYEFIKNHHGEK
  VELAFDKPVEVLSVEAYEKLVHESYSHNRSKMKKLLMEDIPDQFIERQLNDSRYISKVVKSLLS
  NIVREENEQEAISKNVIPCTGGITDRLKKDWGINDVWNKIVLPRFIRLNELTESTRFTSINTNN
  TMIPSMPLELQKGFNKKRIDHRHHAMDAIIIACANRNIVNYLNNVSASKNTKITRRDLQTLLCH
  KDKTDNNGNYKWVIDKPWETFTQDTLTALQKITVSFKQNLRVINKTTNHYQHYENGKKIVSNQS
  KGDSWAIRKSMHKETVHGEVNLRMIKTVSFNEALKKPQAIVEMDLKKKILAMLELGYDTKRIKN
  YFEENKDTWQDINPSKIKVYYFTKETKDRYFAVRKPIDTSFDKKKIKESITDTGIQQIMLRHLE
  TKDNDPTLAFSPDGIDEMNRNILILNKGKKHQPIYKVRVYEKAEKFTVGQKGNKRTKFVEAAKG
  TNLFFAIYETEEIDKDTKKVIRKRSYSTIPLNVVIERQKQGLSSAPEDENGNLPKYILSPNDLV
  YVPTQEEINKGEVVMPIDRDRIYKMVDSSGITANFIPASTANLIFALPKATAEIYCNGENCIQN
  EYGIGSPQSKNQKAITGEMVKEICFPIKVDRLGNIIQVGSCILTN

  SEQ ID NO: 338

  MSRSLTFSFDIGYASIGWAVIASASHDDADPSVCGCGTVLFPKDDCQAFKRREYRRLRRNIRSR
  RVRIERIGRLLVQAQIITPEMKETSGHPAPFYLASEALKGHRTLAPIELWHVLRWYAHNRGYDN
  NASWSNSLSEDGGNGEDTERVKHAQDLMDKHGTATMAETICRELKLEEGKADAPMEVSTPAYKN
  LNTAFPRLIVEKEVRRILELSAPLIPGLTAEIIELIAQHHPLTTEQRGVLLQHGIKLARRYRGS
  LLFGQLIPRFDNRIISRCPVTWAQVYEAELKKGNSEQSARERAEKLSKVPTANCPEFYEYRMAR
  ILCNIRADGEPLSAEIRRELMNQARQEGKLTKASLEKAISSRLGKETETNVSNYFTLHPDSEEA
  LYLNPAVEVLQRSGIGQILSPSVYRIAANRLRRGKSVTPNYLLNLLKSRGESGEALEKKIEKES
  KKKEADYADTPLKPKYATGRAPYARTVLKKVVEEILDGEDPTRPARGEAHPDGELKAHDGCLYC
  LLDTDSSVNQHQKERRLDTMTNNHLVRHRMLILDRLLKDLIQDFADGQKDRISRVCVEVGKELT
  TFSAMDSKKIQRELTLRQKSHTDAVNRLKRKLPGKALSANLIRKCRIAMDMNWTCPFTGATYGD
  HELENLELEHIVPHSFRQSNALSSLVLTWPGVNRMKGQRTGYDFVEQEQENPVPDKPNLHICSL
  NNYRELVEKLDDKKGHEDDRRRKKKRKALLMVRGLSHKHQSQNHEAMKEIGMTEGMMTQSSHLM
  KLACKSIKTSLPDAHIDMIPGAVTAEVRKAWDVFGVFKELCPEAADPDSGKILKENLRSLTHLH
  HALDACVLGLIPYIIPAHHNGLLRRVLAMRRIPEKLIPQVRPVANQRHYVLNDDGRMMLRDLSA
  SLKENIREQLMEQRVIQHVPADMGGALLKETMQRVLSVDGSGEDAMVSLSKKKDGKKEKNQVKA
  SKLVGVFPEGPSKLKALKAAIEIDGNYGVALDPKPVVIRHIKVFKRIMALKEQNGGKPVRILKK
  GMLIHLTSSKDPKHAGVWRIESIQDSKGGVKLDLQRAHCAVPKNKTHECNWREVDLISLLKKYQ
  MKRYPTSYTGTPR

  SEQ ID NO: 339

  MTQKVLGLDLGTNSIGSAVRNLDLSDDLQWQLEFFSSDIFRSSVNKESNGREYSLAAQRSAHRR
  SRGLNEVRRRRLWATLNLLIKHGFCPMSSESLMRWCTYDKRKGLFREYPIDDKDFNAWILLDFN
  GDGRPDYSSPYQLRRELVTRQFDFEQPIERYKLGRALYHIAQHRGFKSSKGETLSQQETNSKPS
  STDEIPDVAGAMKASEEKLSKGLSTYMKEHNLLTVGAAFAQLEDEGVRVRNNNDYRAIRSQFQH
  EIETIFKFQQGLSVESELYERLISEKKNVGTIFYKRPLRSQRGNVGKCTLERSKPRCAIGHPLF
  EKFRAWTLINNIKVRMSVDTLDEQLPMKLRLDLYNECFLAFVRTEFKFEDIRKYLEKRLGIHFS
  YNDKTINYKDSTSVAGCPITARFRKMLGEEWESFRVEGQKERQAHSKNNISFHRVSYSIEDIWH
  FCYDAEEPEAVLAFAQETLRLERKKAEELVRIWSAMPQGYAMLSQKAIRNINKILMLGLKYSDA
  VILAKVPELVDVSDEELLSIAKDYYLVEAQVNYDKRINSIVNGLIAKYKSVSEEYRFADHNYEY
  LLDESDEKDIIRQIENSLGARRWSLMDANEQTDILQKVRDRYQDFFRSHERKFVESPKLGESFE
  NYLTKKFPMVEREQWKKLYHPSQITIYRPVSVGKDRSVLRLGNPDIGAIKNPTVLRVLNTLRRR
  VNQLLDDGVISPDETRVVVETARELNDANRKWALDTYNRIRHDENEKIKKILEEFYPKRDGIST
  DDIDKARYVIDQREVDYFTGSKTYNKDIKKYKFWLEQGGQCMYTGRTINLSNLFDPNAFDIEHT
  IPESLSFDSSDMNLTLCDAHYNRFIKKNHIPTDMPNYDKAITIDGKEYPAITSQLQRWVERVER
  LNRNVEYWKGQARRAQNKDRKDQCMREMHLWKMELEYWKKKLERFTVTEVTDGFKNSQLVDTRV
  ITRHAVLYLKSIFPHVDVQRGDVTAKFRKILGIQSVDEKKDRSLHSHHAIDATTLTIIPVSAKR
  DRMLELFAKIEEINKMLSFSGSEDRTGLIQELEGLKNKLQMEVKVCRIGHNVSEIGTFINDNII
  VNHHIKNQALTPVRRRLRKKGYIVGGVDNPRWQTGDALRGEIHKASYYGAITQFAKDDEGKVLM
  KEGRPQVNPTIKFVIRRELKYKKSAADSGFASWDDLGKAIVDKELFALMKGQFPAETSFKDACE
  QGIYMIKKGKNGMPDIKLHHIRHVRCEAPQSGLKIKEQTYKSEKEYKRYFYAAVGDLYAMCCYT
  NGKIREFRIYSLYDVSCHRKSDIEDIPEFITDKKGNRLMLDYKLRTGDMILLYKDNPAELYDLD
  NVNLSRRLYKINRFESQSNLVLMTHHLSTSKERGRSLGKTVDYQNLPESIRSSVKSLNFLIMGE
  NRDFVIKNGKIIFNHR

  SEQ ID NO: 340

  MLVSPISVDLGGKNTGFFSFTDSLDNSQSGTVIYDESFVLSQVGRRSKRHSKRNNLRNKLVKRL
  FLLILQEHHGLSIDVLPDEIRGLFNKRGYTYAGFELDEKKKDALESDTLKEFLSEKLQSIDRDS
  DVEDFLNQIASNAESFKDYKKGFEAVFASATHSPNKKLELKDELKSEYGENAKELLAGLRVTKE
  ILDEFDKQENQGNLPRAKYFEELGEYIATNEKVKSFFDSNSLKLTDMTKLIGNISNYQLKELRR
  YFNDKEMEKGDIWIPNKLHKITERFVRSWHPKNDADRQRRAELMKDLKSKEIMELLTTTEPVMT
  IPPYDDMNNRGAVKCQTLRLNEEYLDKHLPNWRDIAKRLNHGKFNDDLADSTVKGYSEDSTLLH
  RLLDTSKEIDIYELRGKKPNELLVKTLGQSDANRLYGFAQNYYELIRQKVRAGIWVPVKNKDDS
  LNLEDNSNMLKRCNHNPPHKKNQIHNLVAGILGVKLDEAKFAEFEKELWSAKVGNKKLSAYCKN
  IEELRKTHGNTFKIDIEELRKKDPAELSKEEKAKLRLTDDVILNEWSQKIANFFDIDDKHRQRF
  NNLFSMAQLHTVIDTPRSGFSSTCKRCTAENRFRSETAFYNDETGEFHKKATATCQRLPADTQR
  PFSGKIERYIDKLGYELAKIKAKELEGMEAKEIKVPIILEQNAFEYEESLRKSKTGSNDRVINS
  KKDRDGKKLAKAKENAEDRLKDKDKRIKAFSSGICPYCGDTIGDDGEIDHILPRSHTLKIYGTV
  FNPEGNLIYVHQKCNQAKADSIYKLSDIKAGVSAQWIEEQVANIKGYKTFSVLSAEQQKAFRYA
  LFLQNDNEAYKKVVDWLRTDQSARVNGTQKYLAKKIQEKLTKMLPNKHLSFEFILADATEVSEL
  RRQYARQNPLLAKAEKQAPSSHAIDAVMAFVARYQKVFKDGTPPNADEVAKLAMLDSWNPASNE
  PLTKGLSTNQKIEKMIKSGDYGQKNMREVFGKSIFGENAIGERYKPIVVQEGGYYIGYPATVKK
  GYELKNCKVVTSKNDIAKLEKIIKNQDLISLKENQYIKIFSINKQTISELSNRYFNMNYKNLVE
  RDKEIVGLLEFIVENCRYYTKKVDVKFAPKYIHETKYPFYDDWRRFDEAWRYLQENQNKTSSKD
  RFVIDKSSLNEYYQPDKNEYKLDVDTQPIWDDFCRWYFLDRYKTANDKKSIRIKARKTFSLLAE
  SGVQGKVFRAKRKIPTGYAYQALPMDNNVIAGDYANILLEANSKTLSLVPKSGISIEKQLDKKL
  DVIKKTDVRGLAIDNNSFFNADFDTHGIRLIVENTSVKVGNFPISAIDKSAKRMIFRALFEKEK
  GKRKKKTTISFKESGPVQDYLKVFLKKIVKIQLRTDGSISNIVVRKNAADFTLSFRSEHIQKLL
  K

  SEQ ID NO: 341

  MAYRLGLDIGITSVGWAVVALEKDESGLKPVRIQDLGVRIFDKAEDSKTGASLALPRREARSAR
  RRTRRRRHRLWRVKRLLEQHGILSMEQIEALYAQRTSSPDVYALRVAGLDRCLIAEEIARVLIH
  IAHRRGFQSNRKSEIKDSDAGKLLKAVQENENLMQSKGYRTVAEMLVSEATKTDAEGKLVHGKK
  HGYVSNVRNKAGEYRHTVSRQAIVDEVRKIFAAQRALGNDVMSEELEDSYLKILCSQRNFDDGP
  GGDSPYGHGSVSPDGVRQSIYERMVGSCTFETGEKRAPRSSYSFERFQLLTKVVNLRIYRQQED
  GGRYPCELTQTERARVIDCAYEQTKITYGKLRKLLDMKDTESFAGLTYGLNRSRNKTEDTVFVE
  MKFYHEVRKALQRAGVFIQDLSIETLDQIGWILSVWKSDDNRRKKLSTLGLSDNVIEELLPLNG
  SKFGHLSLKAIRKILPFLEDGYSYDVACELAGYQFQGKTEYVKQRLLPPLGEGEVTNPVVRRAL
  SQAIKVVNAVIRKHGSPESIHIELARELSKNLDERRKIEKAQKENQKNNEQIKDEIREILGSAH
  VTGRDIVKYKLFKQQQEFCMYSGEKLDVTRLFEPGYAEVDHIIPYGISFDDSYDNKVLVKTEQN
  RQKGNRTPLEYLRDKPEQKAKFIALVESIPLSQKKKNHLLMDKRAIDLEQEGFRERNLSDTRYI
  TRALMNHIQAWLLFDETASTRSKRVVCVNGAVTAYMRARWGLTKDRDAGDKHHAADAVVVACIG
  DSLIQRVTKYDKFKRNALADRNRYVQQVSKSEGITQYVDKETGEVFTWESFDERKFLPNEPLEP
  WPFFRDELLARLSDDPSKNIRAIGLLTYSETEQIDPIFVSRMPTRKVTGAAHKETIRSPRIVKV
  DDNKGTEIQVVVSKVALTELKLTKDGEIKDYFRPEDDPRLYNTLRERLVQFGGDAKAAFKEPVY
  KISKDGSVRTPVRKVKIQEKLTLGVPVHGGRGIAENGGMVRIDVFAKGGKYYFVPIYVADVLKR
  ELPNRLATAHKPYSEWRVVDDSYQFKFSLYPNDAVMIKPSREVDITYKDRKEPVGCRIMYFVSA
  NIASASISLRTHDNSGELEGLGIQGLEVFEKYVVGPLGDTHPVYKERRMPFRVERKMN

  SEQ ID NO: 342

  MPVLSPLSPNAAQGRRRWSLALDIGEGSIGWAVAEVDAEGRVLQLTGTGVTLFPSAWSNENGTY
  VAHGAADRAVRGQQQRHDSRRRRLAGLARLCAPVLERSPEDLKDLTRTPPKADPRAIFFLRADA
  ARRPLDGPELFRVLHHMAAHRGIRLAELQEVDPPPESDADDAAPAATEDEDGTRRAAADERAFR
  RLMAEHMHRHGTQPTCGEIMAGRLRETPAGAQPVTRARDGLRVGGGVAVPTRALIEQEFDAIRA
  IQAPRHPDLPWDSLRRLVLDQAPIAVPPATPCLFLEELRRRGETFQGRTITREAIDRGLTVDPL
  IQALRIRETVGNLRLHERITEPDGRQRYVPRAMPELGLSHGELTAPERDTLVRALMHDPDGLAA
  KDGRIPYTRLRKLIGYDNSPVCFAQERDTSGGGITVNPTDPLMARWIDGWVDLPLKARSLYVRD
  VVARGADSAALARLLAEGAHGVPPVAAAAVPAATAAILESDIMQPGRYSVCPWAAEAILDAWAN
  APTEGFYDVTRGLFGFAPGEIVLEDLRRARGALLAHLPRTMAAARTPNRAAQQRGPLPAYESVI
  PSQLITSLRRAHKGRAADWSAADPEERNPFLRTWTGNAATDHILNQVRKTANEVITKYGNRRGW
  DPLPSRITVELAREAKHGVIRRNEIAKENRENEGRRKKESAALDTFCQDNTVSWQAGGLPKERA
  ALRLRLAQRQEFFCPYCAERPKLRATDLFSPAETEIDHVIERRMGGDGPDNLVLAHKDCNNAKG
  KKTPHEHAGDLLDSPALAALWQGWRKENADRLKGKGHKARTPREDKDFMDRVGWRFEEDARAKA
  EENQERRGRRMLHDTARATRLARLYLAAAVMPEDPAEIGAPPVETPPSPEDPTGYTAIYRTISR
  VQPVNGSVTHMLRQRLLQRDKNRDYQTHHAEDACLLLLAGPAVVQAFNTEAAQHGADAPDDRPV
  DLMPTSDAYHQQRRARALGRVPLATVDAALADIVMPESDRQDPETGRVHWRLTRAGRGLKRRID
  DLTRNCVILSRPRRPSETGTPGALHNATHYGRREITVDGRTDTVVTQRMNARDLVALLDNAKIV
  PAARLDAAAPGDTILKEICTEIADRHDRVVDPEGTHARRWISARLAALVPAHAEAVARDIAELA
  DLDALADADRTPEQEARRSALRQSPYLGRAISAKKADGRARAREQEILTRALLDPHWGPRGLRH
  LIMREARAPSLVRIRANKTDAFGRPVPDAAVWVKTDGNAVSQLWRLTSVVTDDGRRIPLPKPIE
  KRIEISNLEYARLNGLDEGAGVTGNNAPPRPLRQDIDRLTPLWRDHGTAPGGYLGTAVGELEDK
  ARSALRGKAMRQTLTDAGITAEAGWRLDSEGAVCDLEVAKGDTVKKDGKTYKVGVITQGIFGMP
  VDAAGSAPRTPEDCEKFEEQYGIKPWKAKGIPLA

  SEQ ID NO: 343

  MNYTEKEKLFMKYILALDIGIASVGWAILDKESETVIEAGSNIFPEASAADNQLRRDMRGAKRN
  NRRLKTRINDFIKLWENNNLSIPQFKSTEIVGLKVRAITEEITLDELYLILYSYLKHRGISYLE
  DALDDTVSGSSAYANGLKLNAKELETHYPCEIQQERLNTIGKYRGQSQIINENGEVLDLSNVFT
  IGAYRKEIQRVFEIQKKYHPELTDEFCDGYMLIFNRKRKYYEGPGNEKSRTDYGRFTTKLDANG
  NYITEDNIFEKLIGKCSVYPDELRAAAASYTAQEYNVLNDLNNLTINGRKLEENEKHEIVERIK
  SSNTINMRKIISDCMGENIDDFAGARIDKSGKEIFHKFEVYNKMRKALLEIGIDISNYSREELD
  EIGYIMTINTDKEAMMEAFQKSWIDLSDDVKQCLINMRKTNGALFNKWQSFSLKIMNELIPEMY
  AQPKEQMTLLTEMGVTKGTQEEFAGLKYIPVDVVSEDIFNPVVRRSVRISFKILNAVLKKYKAL
  DTIVIEMPRDRNSEEQKKRINDSQKLNEKEMEYIEKKLAVTYGIKLSPSDFSSQKQLSLKLKLW
  NEQDGICLYSGKTIDPNDIINNPQLFEIDHIIPRSISFDDARSNKVLVYRSENQKKGNQTPYYY
  LTHSHSEWSFEQYKATVMNLSKKKEYAISRKKIQNLLYSEDITKMDVLKGFINRNINDTSYASR
  LVLNTIQNFFMANEADTKVKVIKGSYTHQMRCNLKLDKNRDESYSHHAVDAMLIGYSELGYEAY
  HKLQGEFIDFETGEILRKDMWDENMSDEVYADYLYGKKWANIRNEVVKAEKNVKYWHYVMRKSN
  RGLCNQTIRGTREYDGKQYKINKLDIRTKEGIKVFAKLAFSKKDSDRERLLVYLNDRRTFDDLC
  KIYEDYSDAANPFVQYEKETGDIIRKYSKKHNGPRIDKLKYKDGEVGACIDISHKYGFEKGSKK
  VILESLVPYRMDVYYKEENHSYYLVGVKQSDIKFEKGRNVIDEEAYARILVNEKMIQPGQSRAD
  LENLGFKFKLSFYKNDIIEYEKDGKIYTERLVSRTMPKQRNYIETKPIDKAKFEKQNLVGLGKT
  KFIKKYRYDILGNKYSCSEEKFTSFC

  SEQ ID NO: 344

  MLRLYCANNLVLNNVQNLWKYLLLLIFDKKIIFLFKIKVILIRRYMENNNKEKIVIGFDLGVAS
  VGWSIVNAETKEVIDLGVRLFSEPEKADYRRAKRTTRRLLRRKKFKREKFHKLILKNAEIFGLQ
  SRNEILNVYKDQSSKYRNILKLKINALKEEIKPSELVWILRDYLQNRGYFYKNEKLTDEFVSNS
  FPSKKLHEHYEKYGFFRGSVKLDNKLDNKKDKAKEKDEEEESDAKKESEELIFSNKQWINEIVK
  VFENQSYLTESFKEEYLKLFNYVRPFNKGPGSKNSRTAYGVFSTDIDPETNKFKDYSNIWDKTI
  GKCSLFEEEIRAPKNLPSALIFNLQNEICTIKNEFTEFKNWWLNAEQKSEILKFVFTELFNWKD
  KKYSDKKFNKNLQDKIKKYLLNFALENFNLNEEILKNRDLENDTVLGLKGVKYYEKSNATADAA
  LEFSSLKPLYVFIKFLKEKKLDLNYLLGLENTEILYFLDSIYLAISYSSDLKERNEWFKKLLKE
  LYPKIKNNNLEIIENVEDIFEITDQEKFESFSKTHSLSREAFNHIIPLLLSNNEGKNYESLKHS
  NEELKKRTEKAELKAQQNQKYLKDNFLKEALVPLSVKTSVLQAIKIFNQIIKNFGKKYEISQVV
  IEMARELTKPNLEKLLNNATNSNIKILKEKLDQTEKFDDFTKKKFIDKIENSVVFRNKLFLWFE
  QDRKDPYTQLDIKINEIEDETEIDHVIPYSKSADDSWFNKLLVKKSTNQLKKNKTVWEYYQNES
  DPEAKWNKFVAWAKRIYLVQKSDKESKDNSEKNSIFKNKKPNLKFKNITKKLFDPYKDLGFLAR
  NLNDTRYATKVFRDQLNNYSKHHSKDDENKLFKVVCMNGSITSFLRKSMWRKNEEQVYRFNFWK
  KDRDQFFHHAVDASIIAIFSLLTKTLYNKLRVYESYDVQRREDGVYLINKETGEVKKADKDYWK
  DQHNFLKIRENAIEIKNVLNNVDFQNQVRYSRKANTKLNTQLFNETLYGVKEFENNFYKLEKVN
  LFSRKDLRKFILEDLNEESEKNKKNENGSRKRILTEKYIVDEILQILENEEFKDSKSDINALNK
  YMDSLPSKFSEFFSQDFINKCKKENSLILTFDAIKHNDPKKVIKIKNLKFFREDATLKNKQAVH
  KDSKNQIKSFYESYKCVGFIWLKNKNDLEESIFVPINSRVIHFGDKDKDIFDFDSYNKEKLLNE
  INLKRPENKKFNSINEIEFVKFVKPGALLLNFENQQIYYISTLESSSLRAKIKLLNKMDKGKAV
  SMKKITNPDEYKIIEHVNPLGINLNWTKKLENNN

  SEQ ID NO: 345

  MLMSKHVLGLDLGVGSIGWCLIALDAQGDPAEILGMGSRVVPLNNATKAIEAFNAGAAFTASQE
  RTARRTMRRGFARYQLRRYRLRRELEKVGMLPDAALIQLPLLELWELRERAATAGRRLTLPELG
  RVLCHINQKRGYRHVKSDAAAIVGDEGEKKKDSNSAYLAGIRANDEKLQAEHKTVGQYFAEQLR
  QNQSESPTGGISYRIKDQIFSRQCYIDEYDQIMAVQRVHYPDILTDEFIRMLRDEVIFMQRPLK
  SCKHLVSLCEFEKQERVMRVQQDDGKGGWQLVERRVKFGPKVAPKSSPLFQLCCIYEAVNNIRL
  TRPNGSPCDITPEERAKIVAHLQSSASLSFAALKKLLKEKALIADQLTSKSGLKGNSTRVALAS
  ALQPYPQYHHLLDMELETRMMTVQLTDEETGEVTEREVAVVTDSYVRKPLYRLWHILYSIEERE
  AMRRALITQLGMKEEDLDGGLLDQLYRLDFVKPGYGNKSAKFICKLLPQLQQGLGYSEACAAVG
  YRHSNSPTSEEITERTLLEKIPLLQRNELRQPLVEKILNQMINLVNALKAEYGIDEVRVELARE
  LKMSREERERMARNNKDREERNKGVAAKIRECGLYPTKPRIQKYMLWKEAGRQCLYCGRSIEEE
  QCLREGGMEVEHIIPKSVLYDDSYGNKTCACRRCNKEKGNRTALEYIRAKGREAEYMKRINDLL
  KEKKISYSKHQRLRWLKEDIPSDFLERQLRLTQYISRQAMAILQQGIRRVSASEGGVTARLRSL
  WGYGKILHTLNLDRYDSMGETERVSREGEATEELHITNWSKRMDHRHHAIDALVVACTRQSYIQ
  RLNRLSSEFGREDKKKEDQEAQEQQATETGRLSNLERWLTQRPHFSVRTVSDKVAEILISYRPG
  QRVVTRGRNIYRKKMADGREVSCVQRGVLVPRGELMEASFYGKILSQGRVRIVKRYPLHDLKGE
  VVDPHLRELITTYNQELKSREKGAPIPPLCLDKDKKQEVRSVRCYAKTLSLDKAIPMCFDEKGE
  PTAFVKSASNHHLALYRTPKGKLVESIVTFWDAVDRARYGIPLVITHPREVMEQVLQRGDIPEQ
  VLSLLPPSDWVFVDSLQQDEMVVIGLSDEELQRALEAQNYRKISEHLYRVQKMSSSYYVFRYHL
  ETSVADDKNTSGRIPKFHRVQSLKAYEERNIRKVRVDLLGRISLL

  SEQ ID NO: 346

  MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRV
  RLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDG
  NSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSE
  ALRILQTQQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILI
  GKCTFYPDEFRAAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF
  KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTE
  REGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMT
  ILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMAR
  ETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERC
  LYTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA
  WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRA
  HKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQ
  LLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRQ
  AKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNK
  QINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQ
  SVSPWRADVYFNKTTGKYEILGLKYADLQFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLY
  KNDLLLVKDTETKEQQLFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKG
  LGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDF

  SEQ ID NO: 347

  MNAEHGKEGLLIMEENFQYRIGLDIGITSVGWAVLQNNSQDEPVRITDLGVRIFDVAENPKNGD
  ALAAPRRDARTTRRRLRRRRHRLERIKFLLQENGLIEMDSFMERYYKGNLPDVYQLRYEGLDRK
  LKDEELAQVLIHIAKHRGFRSTRKAETKEKEGGAVLKATTENQKIMQEKGYRTVGEMLYLDEAF
  HTECLWNEKGYVLTPRNRPDDYKHTILRSMLVEEVHAIFAAQRAHGNQKATEGLEEAYVEIMTS
  QRSFDMGPGLQPDGKPSPYAMEGFGDRVGKCTFEKDEYRAPKATYTAELFVALQKINHTKLIDE
  FGTGRFFSEEERKTIIGLLLSSKELKYGTIRKKLNIDPSLKFNSLNYSAKKEGETEEERVLDTE
  KAKFASMFWTYEYSKCLKDRTEEMPVGEKADLFDRIGEILTAYKNDDSRSSRLKELGLSGEEID
  GLLDLSPAKYQRVSLKAMRKMQPYLEDGLIYDKACEAAGYDFRALNDGNKKHLLKGEEINAIVN
  DITNPVVKRSVSQTIKVINAIIQKYGSPQAVNIELAREMSKNFQDRTNLEKEMKKRQQENERAK
  QQIIELGKQNPTGQDILKYRLWNDQGGYCLYSGKKIPLEELFDGGYDIDHILPYSITFDDSYRN
  KVLVTAQENRQKGNRTPYEYFGADEKRWEDYEASVRLLVRDYKKQQKLLKKNFTEEERKEFKER
  NLNDTKYITRVVYNMIRQNLELEPFNHPEKKKQVWAVNGAVTSYLRKRWGLMQKDRSTDRHHAM
  DAVVIACCTDGMIHKISRYMQGRELAYSRNFKFPDEETGEILNRDNFTREQWDEKFGVKVPLPW
  NSFRDELDIRLLNEDPKNFLLTHADVQRELDYPGWMYGEEESPIEEGRYINYIRPLFVSRMPNH
  KVTGSAHDATIRSARDYETRGVVITKVPLTDLKLNKDNEIEGYYDKDSDRLLYQALVRQLLLHG
  NDGKKAFAEDFHKPKADGTEGPVVRKVKIEKKQTSGVMVRGGTGIAANGEMVRIDVFRENGKYY
  FVPVYTADVVRKVLPNRAATHTKPYSEWRVMDDANFVFSLYSRDLIHVKSKKDIKTNLVNGGLL
  LQKEIFAYYTGADIATASIAGFANDSNFKFRGLGIQSLEIFEKCQVDILGNISVVRHENRQEFH

  SEQ ID NO: 348

  MRVLGLDAGIASLGWALIEIEESNRGELSQGTIIGAGTWMFDAPEEKTQAGAKLKSEQRRTFRG
  QRRVVRRRRQRMNEVRRILHSHGLLPSSDRDALKQPGLDPWRIRAEALDRLLGPVELAVALGHI
  ARHRGFKSNSKGAKTNDPADDTSKMKRAVNETREKLARFGSAAKMLVEDESFVLRQTPTKNGAS
  EIVRRFRNREGDYSRSLLRDDLAAEMRALFTAQARFQSAIATADLQTAFTKAAFFQRPLQDSEK
  LVGPCPFEVDEKRAPKRGYSFELFRFLSRLNHVTLRDGKQERTLTRDELALAAADFGAAAKVSF
  TALRKKLKLPETTVFVGVKADEESKLDVVARSGKAAEGTARLRSVIVDALGELAWGALLCSPEK
  LDKIAEVISFRSDIGRISEGLAQAGCNAPLVDALTAAASDGRFDPFTGAGHISSKAARNILSGL
  RQGMTYDKACCAADYDHTASRERGAFDVGGHGREALKRILQEERISRELVGSPTARKALIESIK
  QVKAIVERYGVPDRIHVELARDVGKSIEEREEITRGIEKRNRQKDKLRGLFEKEVGRPPQDGAR
  GKEELLRFELWSEQMGRCLYTDDYISPSQLVATDDAVQVDHILPWSRFADDSYANKTLCMAKAN
  QDKKGRTPYEWFKAEKTDTEWDAFIVRVEALADMKGFKKRNYKLRNAEEAAAKFRNRNLNDTRW
  ACRLLAEALKQLYPKGEKDKDGKERRRVFSRPGALTDRLRRAWGLQWMKKSTKGDRIPDDRHHA
  LDAIVIAATTESLLQRATREVQEIEDKGLHYDLVKNVTPPWPGFREQAVEAVEKVFVARAERRR
  ARGKAHDATIRHIAVREGEQRVYERRKVAELKLADLDRVKDAERNARLIEKLRNWIEAGSPKDD
  PPLSPKGDPIFKVRLVTKSKVNIALDTGNPKRPGTVDRGEMARVDVFRKASKKGKYEYYLVPIY
  PHDIATMKTPPIRAVQAYKPEDEWPEMDSSYEFCWSLVPMTYLQVISSKGEIFEGYYRGMNRSV
  GAIQLSAHSNSSDVVQGIGARTLTEFKKFNVDRFGRKHEVERELRTWRGETWRGKAYI

  SEQ ID NO: 349

  MGNYYLGLDVGIGSIGWAVINIEKKRIEDFNVRIFKSGEIQEKNRNSRASQQCRRSRGLRRLYR
  RKSHRKLRLKNYLSIIGLTTSEKIDYYYETADNNVIQLRNKGLSEKLTPEEIAACLIHICNNRG
  YKDFYEVNVEDIEDPDERNEYKEEHDSIVLISNLMNEGGYCTPAEMICNCREFDEPNSVYRKFH
  NSAASKNHYLITRHMLVKEVDLILENQSKYYGILDDKTIAKIKDIIFAQRDFEIGPGKNERFRR
  FTGYLDSIGKCQFFKDQERGSRFTVIADIYAFVNVLSQYTYTNNRGESVFDTSFANDLINSALK
  NGSMDKRELKAIAKSYHIDISDKNSDTSLTKCFKYIKVVKPLFEKYGYDWDKLIENYTDTDNNV
  LNRIGIVLSQAQTPKRRREKLKALNIGLDDGLINELTKLKLSGTANVSYKYMQGSIEAFCEGDL
  YGKYQAKFNKEIPDIDENAKPQKLPPFKNEDDCEFFKNPVVFRSINETRKLINAIIDKYGYPAA
  VNIETADELNKTFEDRAIDTKRNNDNQKENDRIVKEIIECIKCDEVHARHLIEKYKLWEAQEGK
  CLYSGETITKEDMLRDKDKLFEVDHIVPYSLILDNTINNKALVYAEENQKKGQRTPLMYMNEAQ
  AADYRVRVNTMFKSKKCSKKKYQYLMLPDLNDQELLGGWRSRNLNDTRYICKYLVNYLRKNLRF
  DRSYESSDEDDLKIRDHYRVFPVKSRFTSMFRRWWLNEKTWGRYDKAELKKLTYLDHAADAIII
  ANCRPEYVVLAGEKLKLNKMYHQAGKRITPEYEQSKKACIDNLYKLFRMDRRTAEKLLSGHGRL
  TPIIPNLSEEVDKRLWDKNIYEQFWKDDKDKKSCEELYRENVASLYKGDPKFASSLSMPVISLK
  PDHKYRGTITGEEAIRVKEIDGKLIKLKRKSISEITAESINSIYTDDKILIDSLKTIFEQADYK
  DVGDYLKKTNQHFFTTSSGKRVNKVTVIEKVPSRWLRKEIDDNNFSLLNDSSYYCIELYKDSKG
  DNNLQGIAMSDIVHDRKTKKLYLKPDFNYPDDYYTHVMYIFPGDYLRIKSTSKKSGEQLKFEGY
  FISVKNVNENSFRFISDNKPCAKDKRVSITKKDIVIKLAVDLMGKVQGENNGKGISCGEPLSLL
  KEKN

  SEQ ID NO: 350

  MLSRQLLGASHLARPVSYSYNVQDNDVHCSYGERCFMRGKRYRIGIDVGLNSVGLAAVEVSDEN
  SPVRLLNAQSVIHDGGVDPQKNKEAITRKNMSGVARRTRRMRRRKRERLHKLDMLLGKFGYPVI
  EPESLDKPFEEWHVRAELATRYIEDDELRRESISIALRHMARHRGWRNPYRQVDSLISDNPYSK
  QYGELKEKAKAYNDDATAAEEESTPAQLVVAMLDAGYAEAPRLRWRTGSKKPDAEGYLPVRLMQ
  EDNANELKQIFRVQRVPADEWKPLFRSVFYAVSPKGSAEQRVGQDPLAPEQARALKASLAFQEY
  RIANVITNLRIKDASAELRKLTVDEKQSIYDQLVSPSSEDITWSDLCDFLGFKRSQLKGVGSLT
  EDGEERISSRPPRLTSVQRIYESDNKIRKPLVAWWKSASDNEHEAMIRLLSNTVDIDKVREDVA
  YASAIEFIDGLDDDALTKLDSVDLPSGRAAYSVETLQKLTRQMLTTDDDLHEARKTLFNVTDSW
  RPPADPIGEPLGNPSVDRVLKNVNRYLMNCQQRWGNPVSVNIEHVRSSFSSVAFARKDKREYEK
  NNEKRSIFRSSLSEQLRADEQMEKVRESDLRRLEAIQRQNGQCLYCGRTITFRTCEMDHIVPRK
  GVGSTNTRTNFAAVCAECNRMKSNTPFAIWARSEDAQTRGVSLAEAKKRVTMFTFNPKSYAPRE
  VKAFKQAVIARLQQTEDDAAIDNRSIESVAWMADELHRRIDWYFNAKQYVNSASIDDAEAETMK
  TTVSVFQGRVTASARRAAGIEGKIHFIGQQSKTRLDRRHHAVDASVIAMMNTAAAQTLMERESL
  RESQRLIGLMPGERSWKEYPYEGTSRYESFHLWLDNMDVLLELLNDALDNDRIAVMQSQRYVLG
  NSIAHDATIHPLEKVPLGSAMSADLIRRASTPALWCALTRLPDYDEKEGLPEDSHREIRVHDTR
  YSADDEMGFFASQAAQIAVQEGSADIGSAIHHARVYRCWKTNAKGVRKYFYGMIRVFQTDLLRA
  CHDDLFTVPLPPQSISMRYGEPRVVQALQSGNAQYLGSLVVGDEIEMDFSSLDVDGQIGEYLQF
  FSQFSGGNLAWKHWVVDGFFNQTQLRIRPRYLAAEGLAKAFSDDVVPDGVQKIVTKQGWLPPVN
  TASKTAVRIVRRNAFGEPRLSSAHHMPCSWQWRHE

  SEQ ID NO: 351

  MYSIGLDLGISSVGWSVIDERTGNVIDLGVRLFSAKNSEKNLERRTNRGGRRLIRRKTNRLKDA
  KKILAAVGFYEDKSLKNSCPYQLRVKGLTEPLSRGEIYKVTLHILKKRGISYLDEVDTEAAKES
  QDYKEQVRKNAQLLTKYTPGQIQLQRLKENNRVKTGINAQGNYQLNVFKVSAYANELATILKTQ
  QAFYPNELTDDWIALFVQPGIAEEAGLIYRKRPYYHGPGNEANNSPYGRWSDFQKTGEPATNIF
  DKLIGKDFQGELRASGLSLSAQQYNLLNDLTNLKIDGEVPLSSEQKEYILTELMTKEFTRFGVN
  DVVKLLGVKKERLSGWRLDKKGKPEIHTLKGYRNWRKIFAEAGIDLATLPTETIDCLAKVLTLN
  TEREGIENTLAFELPELSESVKLLVLDRYKELSQSISTQSWHRFSLKTLHLLIPELMNATSEQN
  TLLEQFQLKSDVRKRYSEYKKLPTKDVLAEIYNPTVNKTVSQAFKVIDALLVKYGKEQIRYITI
  EMPRDDNEEDEKKRIKELHAKNSQRKNDSQSYFMQKSGWSQEKFQTTIQKNRRFLAKLLYYYEQ
  DGICAYTGLPISPELLVSDSTEIDHIIPISISLDDSINNKVLVLSKANQVKGQQTPYDAWMDGS
  FKKINGKFSNWDDYQKWVESRHFSHKKENNLLETRNIFDSEQVEKFLARNLNDTRYASRLVLNT
  LQSFFTNQETKVRVVNGSFTHTLRKKWGADLDKTRETHHHHAVDATLCAVTSFVKVSRYHYAVK
  EETGEKVMREIDFETGEIVNEMSYWEFKKSKKYERKTYQVKWPNFREQLKPVNLHPRIKFSHQV
  DRKANRKLSDATIYSVREKTEVKTLKSGKQKITTDEYTIGKIKDIYTLDGWEAFKKKQDKLLMK
  DLDEKTYERLLSIAETTPDFQEVEEKNGKVKRVKRSPFAVYCEENDIPAIQKYAKKNNGPLIRS
  LKYYDGKLNKHINITKDSQGRPVEKTKNGRKVTLQSLKPYRYDIYQDLETKAYYTVQLYYSDLR
  FVEGKYGITEKEYMKKVAEQTKGQVVRFCFSLQKNDGLEIEWKDSQRYDVRFYNFQSANSINFK
  GLEQEMMPAENQFKQKPYNNGAINLNIAKYGKEGKKLRKFNTDILGKKHYLFYEKEPKNIIK

  SEQ ID NO: 352

  MYFYKNKENKLNKKVVLGLDLGIASVGWCLTDISQKEDNKFPIILHGVRLFETVDDSDDKLLNE
  TRRKKRGQRRRNRRLFTRKRDFIKYLIDNNIIELEFDKNPKILVRNFIEKYINPFSKNLELKYK
  SVTNLPIGFHNLRKAAINEKYKLDKSELIVLLYFYLSLRGAFFDNPEDTKSKEMNKNEIEIFDK
  NESIKNAEFPIDKIIEFYKISGKIRSTINLKFGHQDYLKEIKQVFEKQNIDFMNYEKFAMEEKS
  FFSRIRNYSEGPGNEKSFSKYGLYANENGNPELIINEKGQKIYTKIFKTLWESKIGKCSYDKKL
  YRAPKNSFSAKVFDITNKLTDWKHKNEYISERLKRKILLSRFLNKDSKSAVEKILKEENIKFEN
  LSEIAYNKDDNKINLPIINAYHSLTTIFKKHLINFENYLISNENDLSKLMSFYKQQSEKLFVPN
  EKGSYEINQNNNVLHIFDAISNILNKFSTIQDRIRILEGYFEFSNLKKDVKSSEIYSEIAKLRE
  FSGTSSLSFGAYYKFIPNLISEGSKNYSTISYEEKALQNQKNNFSHSNLFEKTWVEDLIASPTV
  KRSLRQTMNLLKEIFKYSEKNNLEIEKIVVEVTRSSNNKHERKKIEGINKYRKEKYEELKKVYD
  LPNENTTLLKKLWLLRQQQGYDAYSLRKIEANDVINKPWNYDIDHIVPRSISFDDSFSNLVIVN
  KLDNAKKSNDLSAKQFIEKIYGIEKLKEAKENWGNWYLRNANGKAFNDKGKFIKLYTIDNLDEF
  DNSDFINRNLSDTSYITNALVNHLTFSNSKYKYSVVSVNGKQTSNLRNQIAFVGIKNNKETERE
  WKRPEGFKSINSNDFLIREEGKNDVKDDVLIKDRSFNGHHAEDAYFITIISQYFRSFKRIERLN
  VNYRKETRELDDLEKNNIKFKEKASFDNFLLINALDELNEKLNQMRFSRMVITKKNTQLFNETL
  YSGKYDKGKNTIKKVEKLNLLDNRTDKIKKIEEFFDEDKLKENELTKLHIFNHDKNLYETLKII
  WNEVKIEIKNKNLNEKNYFKYFVNKKLQEGKISFNEWVPILDNDFKIIRKIRYIKFSSEEKETD
  EIIFSQSNFLKIDQRQNFSFHNTLYWVQIWVYKNQKDQYCFISIDARNSKFEKDEIKINYEKLK
  TQKEKLQIINEEPILKINKGDLFENEEKELFYIVGRDEKPQKLEIKYILGKKIKDQKQIQKPVK
  KYFPNWKKVNLTYMGEIFKK

  SEQ ID NO: 353

  MDNKNYRIGIDVGLNSIGFCAVEVDQHDTPLGFLNLSVYRHDAGIDPNGKKTNTTRLAMSGVAR
  RTRRLFRKRKRRLAALDRFIEAQGWTLPDHADYKDPYTPWLVRAELAQTPIRDENDLHEKLAIA
  VRHIARHRGWRSPWVPVRSLHVEQPPSDQYLALKERVEAKTLLQMPEGATPAEMVVALDLSVDV
  NLRPKNREKTDTRPENKKPGFLGGKLMQSDNANELRKIAKIQGLDDALLRELIELVFAADSPKG
  ASGELVGYDVLPGQHGKRRAEKAHPAFQRYRIASIVSNLRIRHLGSGADERLDVETQKRVFEYL
  LNAKPTADITWSDVAEEIGVERNLLMGTATQTADGERASAKPPVDVTNVAFATCKIKPLKEWWL
  NADYEARCVMVSALSHAEKLTEGTAAEVEVAEFLQNLSDEDNEKLDSFSLPIGRAAYSVDSLER
  LTKRMIENGEDLFEARVNEFGVSEDWRPPAEPIGARVGNPAVDRVLKAVNRYLMAAEAEWGAPL
  SVNIEHVREGFISKRQAVEIDRENQKRYQRNQAVRSQIADHINATSGVRGSDVTRYLAIQRQNG
  ECLYCGTAITFVNSEMDHIVPRAGLGSTNTRDNLVATCERCNKSKSNKPFAVWAAECGIPGVSV
  AEALKRVDFWIADGFASSKEHRELQKGVKDRLKRKVSDPEIDNRSMESVAWMARELAHRVQYYF
  DEKHTGTKVRVFRGSLTSAARKASGFESRVNFIGGNGKTRLDRRHHAMDAATVAMLRNSVAKTL
  VLRGNIRASERAIGAAETWKSFRGENVADRQIFESWSENMRVLVEKFNLALYNDEVSIFSSLRL
  QLGNGKAHDDTITKLQMHKVGDAWSLTEIDRASTPALWCALTRQPDFTWKDGLPANEDRTIIVN
  GTHYGPLDKVGIFGKAAASLLVRGGSVDIGSAIHHARIYRIAGKKPTYGMVRVFAPDLLRYRNE
  DLFNVELPPQSVSMRYAEPKVREAIREGKAEYLGWLVVGDELLLDLSSETSGQIAELQQDFPGT
  THWTVAGFFSPSRLRLRPVYLAQEGLGEDVSEGSKSIIAGQGWRPAVNKVFGSAMPEVIRRDGL
  GRKRRFSYSGLPVSWQG

  SEQ ID NO: 354

  MRLGLDIGTSSIGWWLYETDGAGSDARITGVVDGGVRIFSDGRDPKSGASLAVDRRAARAMRRR
  RDRYLRRRATLMKVLAETGLMPADPAEAKALEALDPFALRAAGLDEPLPLPHLGRALFHLNQRR
  GFKSNRKTDRGDNESGKIKDATARLDMEMMANGARTYGEFLHKRRQKATDPRHVPSVRTRLSIA
  NRGGPDGKEEAGYDFYPDRRHLEEEFHKLWAAQGAHHPELTETLRDLLFEKIFFQRPLKEPEVG
  LCLFSGHHGVPPKDPRLPKAHPLTQRRVLYETVNQLRVTADGREARPLTREERDQVIHALDNKK
  PTKSLSSMVLKLPALAKVLKLRDGERFTLETGVRDAIACDPLRASPAHPDRFGPRWSILDADAQ
  WEVISRIRRVQSDAEHAALVDWLTEAHGLDRAHAEATAHAPLPDGYGRLGLTATTRILYQLTAD
  VVTYADAVKACGWHHSDGRTGECFDRLPYYGEVLERHVIPGSYHPDDDDITRFGRITNPTVHIG
  LNQLRRLVNRIIETHGKPHQIVVELARDLKKSEEQKRADIKRIRDTTEAAKKRSEKLEELEIED
  NGRNRMLLRLWEDLNPDDAMRRFCPYTGTRISAAMIFDGSCDVDHILPYSRTLDDSFPNRTLCL
  REANRQKRNQTPWQAWGDTPHWHAIAANLKNLPENKRWRFAPDAMTRFEGENGFLDRALKDTQY
  LARISRSYLDTLFTKGGHVWVVPGRFTEMLRRHWGLNSLLSDAGRGAVKAKNRTDHRHHAIDAA
  VIAATDPGLLNRISRAAGQGEAAGQSAELIARDTPPPWEGFRDDLRVRLDRIIVSHRADHGRID
  HAARKQGRDSTAGQLHQETAYSIVDDIHVASRTDLLSLKPAQLLDEPGRSGQVRDPQLRKALRV
  ATGGKTGKDFENALRYFASKPGPYQAIRRVRIIKPLQAQARVPVPAQDPIKAYQGGSNHLFEIW
  RLPDGEIEAQVITSFEAHTLEGEKRPHPAAKRLLRVHKGDMVALERDGRRVVGHVQKMDIANGL
  FIVPHNEANADTRNNDKSDPFKWIQIGARPAIASGIRRVSVDEIGRLRDGGTRPI

  SEQ ID NO: 355

  MLHCIAVIRVPPSEEPGFFETHADSCALCHHGCMTYAANDKAIRYRVGIDVGLRSIGFCAVEVD
  DEDHPIRILNSVVHVHDAGTGGPGETESLRKRSGVAARARRRGRAEKQRLKKLDVLLEELGWGV
  SSNELLDSHAPWHIRKRLVSEYIEDETERRQCLSVAMAHIARHRGWRNSFSKVDTLLLEQAPSD
  RMQGLKERVEDRTGLQFSEEVTQGELVATLLEHDGDVTIRGFVRKGGKATKVHGVLEGKYMQSD
  LVAELRQICRTQRVSETTFEKLVLSIFHSKEPAPSAARQRERVGLDELQLALDPAAKQPRAERA
  HPAFQKFKVVATLANMRIREQSAGERSLTSEELNRVARYLLNHTESESPTWDDVARKLEVPRHR
  LRGSSRASLETGGGLTYPPVDDTTVRVMSAEVDWLADWWDCANDESRGHMIDAISNGCGSEPDD
  VEDEEVNELISSATAEDMLKLELLAKKLPSGRVAYSLKTLREVTAAILETGDDLSQAITRLYGV
  DPGWVPTPAPIEAPVGNPSVDRVLKQVARWLKFASKRWGVPQTVNIEHTREGLKSASLLEEERE
  RWERFEARREIRQKEMYKRLGISGPFRRSDQVRYEILDLQDCACLYCGNEINFQTFEVDHIIPR
  VDASSDSRRTNLAAVCHSCNSAKGGLAFGQWVKRGDCPSGVSLENAIKRVRSWSKDRLGLTEKA
  MGKRKSEVISRLKTEMPYEEFDGRSMESVAWMAIELKKRIEGYFNSDRPEGCAAVQVNAYSGRL
  TACARRAAHVDKRVRLIRLKGDDGHHKNRFDRRNHAMDALVIALMTPAIARTIAVREDRREAQQ
  LTRAFESWKNFLGSEERMQDRWESWIGDVEYACDRLNELIDADKIPVTENLRLRNSGKLHADQP
  ESLKKARRGSKRPRPQRYVLGDALPADVINRVTDPGLWTALVRAPGFDSQLGLPADLNRGLKLR
  GKRISADFPIDYFPTDSPALAVQGGYVGLEFHHARLYRIIGPKEKVKYALLRVCAIDLCGIDCD
  DLFEVELKPSSISMRTADAKLKEAMGNGSAKQIGWLVLGDEIQIDPTKFPKQSIGKFLKECGPV
  SSWRVSALDTPSKITLKPRLLSNEPLLKTSRVGGHESDLVVAECVEKIMKKTGWVVEINALCQS
  GLIRVIRRNALGEVRTSPKSGLPISLNLR

  SEQ ID NO: 356

  MRYRVGLDLGTASVGAAVFSMDEQGNPMELIWHYERLFSEPLVPDMGQLKPKKAARRLARQQRR
  QIDRRASRLRRIAIVSRRLGIAPGRNDSGVHGNDVPTLRAMAVNERIELGQLRAVLLRMGKKRG
  YGGTFKAVRKVGEAGEVASGASRLEEEMVALASVQNKDSVTVGEYLAARVEHGLPSKLKVAANN
  EYYAPEYALFRQYLGLPAIKGRPDCLPNMYALRHQIEHEFERIWATQSQFHDVMKDHGVKEEIR
  NAIFFQRPLKSPADKVGRCSLQTNLPRAPRAQIAAQNFRIEKQMADLRWGMGRRAEMLNDHQKA
  VIRELLNQQKELSFRKIYKELERAGCPGPEGKGLNMDRAALGGRDDLSGNTTLAAWRKLGLEDR
  WQELDEVTQIQVINFLADLGSPEQLDTDDWSCRFMGKNGRPRNFSDEFVAFMNELRMTDGFDRL
  SKMGFEGGRSSYSIKALKALTEWMIAPHWRETPETHRVDEEAAIRECYPESLATPAQGGRQSKL
  EPPPLTGNEVVDVALRQVRHTINMMIDDLGSVPAQIVVEMAREMKGGVTRRNDIEKQNKRFASE
  RKKAAQSIEENGKTPTPARILRYQLWIEQGHQCPYCESNISLEQALSGAYTNFEHILPRTLTQI
  GRKRSELVLAHRECNDEKGNRTPYQAFGHDDRRWRIVEQRANALPKKSSRKTRLLLLKDFEGEA
  LTDESIDEFADRQLHESSWLAKVTTQWLSSLGSDVYVSRGSLTAELRRRWGLDTVIPQVRFESG
  MPVVDEEGAEITPEEFEKFRLQWEGHRVTREMRTDRRPDKRIDHRHHLVDAIVTALTSRSLYQQ
  YAKAWKVADEKQRHGRVDVKVELPMPILTIRDIALEAVRSVRISHKPDRYPDGRFFEATAYGIA
  QRLDERSGEKVDWLVSRKSLTDLAPEKKSIDVDKVRANISRIVGEAIRLHISNIFEKRVSKGMT
  PQQALREPIEFQGNILRKVRCFYSKADDCVRIEHSSRRGHHYKMLLNDGFAYMEVPCKEGILYG
  VPNLVRPSEAVGIKRAPESGDFIRFYKGDTVKNIKTGRVYTIKQILGDGGGKLILTPVTETKPA
  DLLSAKWGRLKVGGRNIHLLRLCAE

  SEQ ID NO: 357

  MIGEHVRGGCLFDDHWTPNWGAFRLPNTVRTFTKAENPKDGSSLAEPRRQARGLRRRLRRKTQR
  LEDLRRLLAKEGVLSLSDLETLFRETPAKDPYQLRAEGLDRPLSFPEWVRVLYHITKHRGFQSN
  RRNPVEDGQERSRQEEEGKLLSGVGENERLLREGGYRTAGEMLARDPKFQDHRRNRAGDYSHTL
  SRSLLLEEARRLFQSQRTLGNPHASSNLEEAFLHLVAFQNPFASGEDIRNKAGHCSLEPDQIRA
  PRRSASAETFMLLQKTGNLRLIHRRTGEERPLTDKEREQIHLLAWKQEKVTHKTLRRHLEIPEE
  WLFTGLPYHRSGDKAEEKLFVHLAGIHEIRKALDKGPDPAVWDTLRSRRDLLDSIADTLTFYKN
  EDEILPRLESLGLSPENARALAPLSFSGTAHLSLSALGKLLPHLEEGKSYTQARADAGYAAPPP
  DRHPKLPPLEEADWRNPVVFRALTQTRKVVNALVRRYGPPWCIHLETARELSQPAKVRRRIETE
  QQANEKKKQQAEREFLDIVGTAPGPGDLLKMRLWREQGGFCPYCEEYLNPTRLAEPGYAEMDHI
  LPYSRSLDNGWHNRVLVHGKDNRDKGNRTPFEAFGGDTARWDRLVAWVQASHLSAPKKRNLLRE
  DFGEEAERELKDRNLTDTRFITKTAATLLRDRLTFHPEAPKDPVMTLNGRLTAFLRKQWGLHKN
  RKNGDLHHALDAAVLAVASRSFVYRLSSHNAAWGELPRGREAENGFSLPYPAFRSEVLARLCPT
  REEILLRLDQGGVGYDEAFRNGLRPVFVSRAPSRRLRGKAHMETLRSPKWKDHPEGPRTASRIP
  LKDLNLEKLERMVGKDRDRKLYEALRERLAAFGGNGKKAFVAPFRKPCRSGEGPLVRSLRIFDS
  GYSGVELRDGGEVYAVADHESMVRVDVYAKKNRFYLVPVYVADVARGIVKNRAIVAHKSEEEWD
  LVDGSFDFRFSLFPGDLVEIEKKDGAYLGYYKSCHRGDGRLLLDRHDRMPRESDCGTFYVSTRK
  DVLSMSKYQVDPLGEIRLVGSEKPPFVL

  SEQ ID NO: 358

  MEKKRKVTLGFDLGIASVGWAIVDSETNQVYKLGSRLFDAPDTNLERRTQRGTRRLLRRRKYRN
  QKFYNLVKRTEVFGLSSREAIENRFRELSIKYPNIIELKTKALSQEVCPDEIAWILHDYLKNRG
  YFYDEKETKEDFDQQTVESMPSYKLNEFYKKYGYFKGALSQPTESEMKDNKDLKEAFFFDFSNK
  EWLKEINYFFNVQKNILSETFIEEFKKIFSFTRDISKGPGSDNMPSPYGIFGEFGDNGQGGRYE
  HIWDKNIGKCSIFTNEQRAPKYLPSALIFNFLNELANIRLYSTDKKNIQPLWKLSSVDKLNILL
  NLFNLPISEKKKKLTSTNINDIVKKESIKSIMISVEDIDMIKDEWAGKEPNVYGVGLSGLNIEE
  SAKENKFKFQDLKILNVLINLLDNVGIKFEFKDRNDIIKNLELLDNLYLFLIYQKESNNKDSSI
  DLFIAKNESLNIENLKLKLKEFLLGAGNEFENHNSKTHSLSKKAIDEILPKLLDNNEGWNLEAI
  KNYDEEIKSQIEDNSSLMAKQDKKYLNDNFLKDAILPPNVKVTFQQAILIFNKIIQKFSKDFEI
  DKVVIELAREMTQDQENDALKGIAKAQKSKKSLVEERLEANNIDKSVFNDKYEKLIYKIFLWIS
  QDFKDPYTGAQISVNEIVNNKVEIDHIIPYSLCFDDSSANKVLVHKQSNQEKSNSLPYEYIKQG
  HSGWNWDEFTKYVKRVFVNNVDSILSKKERLKKSENLLTASYDGYDKLGFLARNLNDTRYATIL
  FRDQLNNYAEHHLIDNKKMFKVIAMNGAVTSFIRKNMSYDNKLRLKDRSDFSHHAYDAAIIALF
  SNKTKTLYNLIDPSLNGIISKRSEGYWVIEDRYTGEIKELKKEDWTSIKNNVQARKIAKEIEEY
  LIDLDDEVFFSRKTKRKTNRQLYNETIYGIATKTDEDGITNYYKKEKFSILDDKDIYLRLLRER
  EKFVINQSNPEVIDQIIEIIESYGKENNIPSRDEAINIKYTKNKINYNLYLKQYMRSLTKSLDQ
  FSEEFINQMIANKTFVLYNPTKNTTRKIKFLRLVNDVKINDIRKNQVINKFNGKNNEPKAFYEN
  INSLGAIVFKNSANNFKTLSINTQIAIFGDKNWDIEDFKTYNMEKIEKYKEIYGIDKTYNFHSF
  IFPGTILLDKQNKEFYYISSIQTVRDIIEIKFLNKIEFKDENKNQDTSKTPKRLMFGIKSIMNN
  YEQVDISPFGINKKIFE

  SEQ ID NO: 359

  MGYRIGLDVGITSTGYAVLKTDKNGLPYKILTLDSVIYPRAENPQTGASLAEPRRIKRGLRRRT
  RRTKFRKQRTQQLFIHSGLLSKPEIEQILATPQAKYSVYELRVAGLDRRLTNSELFRVLYFFIG
  HRGFKSNRKAELNPENEADKKQMGQLLNSIEEIRKAIAEKGYRTVGELYLKDPKYNDHKRNKGY
  IDGYLSTPNRQMLVDEIKQILDKQRELGNEKLTDEFYATYLLGDENRAGIFQAQRDFDEGPGAG
  PYAGDQIKKMVGKDIFEPTEDRAAKATYTFQYFNLLQKMTSLNYQNTTGDTWHTLNGLDRQAII
  DAVFAKAEKPTKTYKPTDFGELRKLLKLPDDARFNLVNYGSLQTQKEIETVEKKTRFVDFKAYH
  DLVKVLPEEMWQSRQLLDHIGTALTLYSSDKRRRRYFAEELNLPAELIEKLLPLNFSKFGHLSI
  KSMQNIIPYLEMGQVYSEATTNTGYDFRKKQISKDTIREEITNPVVRRAVTKTIKIVEQIIRRY
  GKPDGINIELARELGRNFKERGDIQKRQDKNRQTNDKIAAELTELGIPVNGQNIIRYKLHKEQN
  GVDPYTGDQIPFERAFSEGYEVDHIIPYSISWDDSYTNKVLTSAKCNREKGNRIPMVYLANNEQ
  RLNALTNIADNIIRNSRKRQKLLKQKLSDEELKDWKQRNINDTRFITRVLYNYFRQAIEFNPEL
  EKKQRVLPLNGEVTSKIRSRWGFLKVREDGDLHHAIDATVIAAITPKFIQQVTKYSQHQEVKNN
  QALWHDAEIKDAEYAAEAQRMDADLFNKIFNGFPLPWPEFLDELLARISDNPVEMMKSRSWNTY
  TPIEIAKLKPVFVVRLANHKISGPAHLDTIRSAKLFDEKGIVLSRVSITKLKINKKGQVATGDG
  IYDPENSNNGDKVVYSAIRQALEAHNGSGELAFPDGYLEYVDHGTKKLVRKVRVAKKVSLPVRL
  KNKAAADNGSMVRIDVFNTGKKFVFVPIYIKDTVEQVLPNKAIARGKSLWYQITESDQFCFSLY
  PGDMVHIESKTGIKPKYSNKENNTSVVPIKNFYGYFDGADIATASILVRAHDSSYTARSIGIAG
  LLKFEKYQVDYFGRYHKVHEKKRQLFVKRDE

  SEQ ID NO: 360

  MQKNINTKQNHIYIKQAQKIKEKLGDKPYRIGLDLGVGSIGFAIVSMEENDGNVLLPKEIIMVG
  SRIFKASAGAADRKLSRGQRNNHRHTRERMRYLWKVLAEQKLALPVPADLDRKENSSEGETSAK
  RFLGDVLQKDIYELRVKSLDERLSLQELGYVLYHIAGHRGSSAIRTFENDSEEAQKENTENKKI
  AGNIKRLMAKKNYRTYGEYLYKEFFENKEKHKREKISNAANNHKFSPTRDLVIKEAEAILKKQA
  GKDGFHKELTEEYIEKLTKAIGYESEKLIPESGFCPYLKDEKRLPASHKLNEERRLWETLNNAR
  YSDPIVDIVTGEITGYYEKQFTKEQKQKLFDYLLTGSELTPAQTKKLLGLKNTNFEDIILQGRD
  KKAQKIKGYKLIKLESMPFWARLSEAQQDSFLYDWNSCPDEKLLTEKLSNEYHLTEEEIDNAFN
  EIVLSSSYAPLGKSAMLIILEKIKNDLSYTEAVEEALKEGKLTKEKQAIKDRLPYYGAVLQEST
  QKIIAKGFSPQFKDKGYKTPHTNKYELEYGRIANPVVHQTLNELRKLVNEIIDILGKKPCEIGL
  ETARELKKSAEDRSKLSREQNDNESNRNRIYEIYIRPQQQVIITRRENPRNYILKFELLEEQKS
  QCPFCGGQISPNDIINNQADIEHLFPIAESEDNGRNNLVISHSACNADKAKRSPWAAFASAAKD
  SKYDYNRILSNVKENIPHKAWRFNQGAFEKFIENKPMAARFKTDNSYISKVAHKYLACLFEKPN
  IICVKGSLTAQLRMAWGLQGLMIPFAKQLITEKESESFNKDVNSNKKIRLDNRHHALDAIVIAY
  ASRGYGNLLNKMAGKDYKINYSERNWLSKILLPPNNIVWENIDADLESFESSVKTALKNAFISV
  KHDHSDNGELVKGTMYKIFYSERGYTLTTYKKLSALKLTDPQKKKTPKDFLETALLKFKGRESE
  MKNEKIKSAIENNKRLFDVIQDNLEKAKKLLEEENEKSKAEGKKEKNINDASIYQKAISLSGDK
  YVQLSKKEPGKFFAISKPTPTTTGYGYDTGDSLCVDLYYDNKGKLCGEIIRKIDAQQKNPLKYK
  EQGFTLFERIYGGDILEVDFDIHSDKNSFRNNTGSAPENRVFIKVGTFTEITNNNIQIWFGNII
  KSTGGQDDSFTINSMQQYNPRKLILSSCGFIKYRSPILKNKEG

  SEQ ID NO: 361

  MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAEVPKTGDSLAMARRL
  ARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWS
  AVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHI
  RNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLG
  HCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQA
  RKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGT
  AFSLFKTDEDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEI
  YGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKS
  FKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
  RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVE
  TSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITN
  LLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQ
  KTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSR
  APNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHK
  DDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYY
  LVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCH
  RGTGNINIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR

  SEQ ID NO: 362

  MQTTNLSYILGLDLGIASVGWAVVEINENEDPIGLIDVGVRIFERAEVPKTGESLALSRRLARS
  TRRLIRRRAHRLLLAKRFLKREGILSTIDLEKGLPNQAWELRVAGLERRLSAIEWGAVLLHLIK
  HRGYLSKRKNESQTNNKELGALLSGVAQNHQLLQSDDYRTPAELALKKFAKEEGHIRNQRGAYT
  HTFNRLDLLAELNLLFAQQHQFGNPHCKEHIQQYMTELLMWQKPALSGEAILKMLGKCTHEKNE
  FKAAKHTYSAERFVWLTKLNNLRILEDGAERALNEEERQLLINHPYEKSKLTYAQVRKLLGLSE
  QAIFKHLRYSKENAESATFMELKAWHAIRKALENQGLKDTWQDLAKKPDLLDEIGTAFSLYKTD
  EDIQQYLTNKVPNSVINALLVSLNFDKFIELSLKSLRKILPLMEQGKRYDQACREIYGHHYGEA
  NQKTSQLLPAIPAQEIRNPVVLRTLSQARKVINAIIRQYGSPARVHIETGRELGKSFKERREIQ
  KQQEDNRTKRESAVQKFKELFSDFSSEPKSKDILKFRLYEQQHGKCLYSGKEINIHRLNEKGYV
  EIDHALPFSRTWDDSFNNKVLVLASENQNKGNQTPYEWLQGKINSERWKNFVALVLGSQCSAAK
  KQRLLTQVIDDNKFIDRNLNDTRYIARFLSNYIQENLLLVGKNKKNVFTPNGQITALLRSRWGL
  IKARENNNRHHALDAIVVACATPSMQQKITRFIRFKEVHPYKIENRYEMVDQESGEIISPHFPE
  PWAYFRQEVNIRVFDNHPDTVLKEMLPDRPQANHQFVQPLFVSRAPTRKMSGQGHMETIKSAKR
  LAEGISVLRIPLTQLKPNLLENMVNKEREPALYAGLKARLAEFNQDPAKAFATPFYKQGGQQVK
  AIRVEQVQKSGVLVRENNGVADNASIVRTDVFIKNNKFFLVPIYTWQVAKGILPNKAIVAHKNE
  DEWEEMDEGAKFKFSLFPNDLVELKTKKEYFFGYYIGLDRATGNISLKEHDGEISKGKDGVYRV
  GVKLALSFEKYQVDELGKNRQICRPQQRQPVR

  SEQ ID NO: 363

  MGIRFAFDLGTNSIGWAVWRTGPGVFGEDTAASLDGSGVLIFKDGRNPKDGQSLATMRRVPRQS
  RKRRDRFVLRRRDLLAALRKAGLFPVDVEEGRRLAATDPYHLRAKALDESLTPHEMGRVIFHLN
  QRRGFRSNRKADRQDREKGKIAEGSKRLAETLAATNCRTLGEFLWSRHRGTPRTRSPTRIRMEG
  EGAKALYAFYPTREMVRAEFERLWTAQSRFAPDLLTPERHEEIAGILFRQRDLAPPKIGCCTFE
  PSERRLPRALPSVEARGIYERLAHLRITTGPVSDRGLTRPERDVLASALLAGKSLTFKAVRKTL
  KILPHALVNFEEAGEKGLDGALTAKLLSKPDHYGAAWHGLSFAEKDTFVGKLLDEADEERLIRR
  LVTENRLSEDAARRCASIPLADGYGRLGRTANTEILAALVEETDETGTVVTYAEAVRRAGERTG
  RNWHHSDERDGVILDRLPYYGEILQRHVVPGSGEPEEKNEAARWGRLANPTVHIGLNQLRKVVN
  RLIAAHGRPDQIVVELARELKLNREQKERLDRENRKNREENERRTAILAEHGQRDTAENKIRLR
  LFEEQARANAGIALCPYTGRAIGIAELFTSEVEIDHILPVSLTLDDSLANRVLCRREANREKRR
  QTPFQAFGATPAWNDIVARAAKLPPNKRWRFDPAALERFEREGGFLGRQLNETKYLSRLAKIYL
  GKICDPDRVYVTPGTLTGLLRARWGLNSILSDSNFKNRSDHRHHAVDAVVIGVLTRGMIQRIAH
  DAARAEDQDLDRVFRDVPVPFEDFRDHVRERVSTITVAVKPEHGKGGALHEDTSYGLVPDTDPN
  AALGNLVVRKPIRSLTAGEVDRVRDRALRARLGALAAPFRDESGRVRDAKGLAQALEAFGAENG
  IRRVRILKPDASVVTIADRRTGVPYRAVAPGENHHVDIVQMRDGSWRGFAASVFEVNRPGWRPE
  WEVKKLGGKLVMRLHKGDMVELSDKDGQRRVKVVQQIEISANRVRLSPHNDGGKLQDRHADADD
  PFRWDLATIPLLKDRGCVAVRVDPIGVVTLRRSNV

  SEQ ID NO: 364

  MMEVFMGRLVLGLDIGITSVGFGIIDLDESEIVDYGVRLFKEGTAAENETRRTKRGGRRLKRRR
  VTRREDMLHLLKQAGIISTSFHPLNNPYDVRVKGLNERLNGEELATALLHLCKHRGSSVETIED
  DEAKAKEAGETKKVLSMNDQLLKSGKYVCEIQKERLRTNGHIRGHENNFKTRAYVDEAFQILSH
  QDLSNELKSAIITIISRKRMYYDGPGGPLSPTPYGRYTYFGQKEPIDLIEKMRGKCSLFPNEPR
  APKLAYSAELFNLLNDLNNLSIEGEKLTSEQKAMILKIVHEKGKITPKQLAKEVGVSLEQIRGF
  RIDTKGSPLLSELTGYKMIREVLEKSNDEHLEDHVFYDEIAEILTKTKDIEGRKKQISELSSDL
  NEESVHQLAGLTKFTAYHSLSFKALRLINEEMLKTELNQMQSITLFGLKQNNELSVKGMKNIQA
  DDTAILSPVAKRAQRETFKVVNRLREIYGEFDSIVVEMAREKNSEEQRKAIRERQKFFEMRNKQ
  VADIIGDDRKINAKLREKLVLYQEQDGKTAYSLEPIDLKLLIDDPNAYEVDHIIPISISLDDSI
  TNKVLVTHRENQEKGNLTPISAFVKGRFTKGSLAQYKAYCLKLKEKNIKTNKGYRKKVEQYLLN
  ENDIYKYDIQKEFINRNLVDTSYASRVVLNTLTTYFKQNEIPTKVFTVKGSLTNAFRRKINLKK
  DRDEDYGHHAIDALIIASMPKMRLLSTIFSRYKIEDIYDESTGEVFSSGDDSMYYDDRYFAFIA
  SLKAIKVRKFSHKIDTKPNRSVADETIYSTRVIDGKEKVVKKYKDIYDPKFTALAEDILNNAYQ
  EKYLMALHDPQTFDQIVKVVNYYFEEMSKSEKYFTKDKKGRIKISGMNPLSLYRDEHGMLKKYS
  KKGDGPAITQMKYFDGVLGNHIDISAHYQVRDKKVVLQQISPYRTDFYYSKENGYKFVTIRYKD
  VRWSEKKKKYVIDQQDYAMKKAEKKIDDTYEFQFSMHRDELIGITKAEGEALIYPDETWHNFNF
  FFHAGETPEILKFTATNNDKSNKIEVKPIHCYCKMRLMPTISKKIVRIDKYATDVVGNLYKVKK
  NTLKFEFD

  SEQ ID NO: 365

  MKKILGVDLGITSFGYAILQETGKDLYRCLDNSVVMRNNPYDEKSGESSQSIRSTQKSMRRLIE
  KRKKRIRCVAQTMERYGILDYSETMKINDPKNNPIKNRWQLRAVDAWKRPLSPQELFAIFAHMA
  KHRGYKSIATEDLIYELELELGLNDPEKESEKKADERRQVYNALRHLEELRKKYGGETIAQTIH
  RAVEAGDLRSYRNHDDYEKMIRREDIEEEIEKVLLRQAELGALGLPEEQVSELIDELKACITDQ
  EMPTIDESLFGKCTFYKDELAAPAYSYLYDLYRLYKKLADLNIDGYEVTQEDREKVIEWVEKKI
  AQGKNLKKITHKDLRKILGLAPEQKIFGVEDERIVKGKKEPRTFVPFFFLADIAKFKELFASIQ
  KHPDALQIFRELAEILQRSKTPQEALDRLRALMAGKGIDTDDRELLELFKNKRSGTRELSHRYI
  LEALPLFLEGYDEKEVQRILGFDDREDYSRYPKSLRHLHLREGNLFEKEENPINNHAVKSLASW
  ALGLIADLSWRYGPFDEIILETTRDALPEKIRKEIDKAMREREKALDKIIGKYKKEFPSIDKRL
  ARKIQLWERQKGLDLYSGKVINLSQLLDGSADIEHIVPQSLGGLSTDYNTIVTLKSVNAAKGNR
  LPGDWLAGNPDYRERIGMLSEKGLIDWKKRKNLLAQSLDEIYTENTHSKGIRATSYLEALVAQV
  LKRYYPFPDPELRKNGIGVRMIPGKVTSKTRSLLGIKSKSRETNFHHAEDALILSTLTRGWQNR
  LHRMLRDNYGKSEAELKELWKKYMPHIEGLTLADYIDEAFRRFMSKGEESLFYRDMFDTIRSIS
  YWVDKKPLSASSHKETVYSSRHEVPTLRKNILEAFDSLNVIKDRHKLTTEEFMKRYDKEIRQKL
  WLHRIGNTNDESYRAVEERATQIAQILTRYQLMDAQNDKEIDEKFQQALKELITSPIEVTGKLL
  RKMRFVYDKLNAMQIDRGLVETDKNMLGIHISKGPNEKLIFRRMDVNNAHELQKERSGILCYLN
  EMLFIFNKKGLIHYGCLRSYLEKGQGSKYIALFNPRFPANPKAQPSKFTSDSKIKQVGIGSATG
  IIKAHLDLDGHVRSYEVFGTLPEGSIEWFKEESGYGRVEDDPHH

  SEQ ID NO: 366

  MRPIEPWILGLDIGTDSLGWAVFSCEEKGPPTAKELLGGGVRLFDSGRDAKDHTSRQAERGAFR
  RARRQTRTWPWRRDRLIALFQAAGLTPPAAETRQIALALRREAVSRPLAPDALWAALLHLAHHR
  GFRSNRIDKRERAAAKALAKAKPAKATAKATAPAKEADDEAGFWEGAEAALRQRMAASGAPTVG
  ALLADDLDRGQPVRMRYNQSDRDGVVAPTRALIAEELAEIVARQSSAYPGLDWPAVTRLVLDQR
  PLRSKGAGPCAFLPGEDRALRALPTVQDFIIRQTLANLRLPSTSADEPRPLTDEEHAKALALLS
  TARFVEWPALRRALGLKRGVKFTAETERNGAKQAARGTAGNLTEAILAPLIPGWSGWDLDRKDR
  VFSDLWAARQDRSALLALIGDPRGPTRVTEDETAEAVADAIQIVLPTGRASLSAKAARAIAQAM
  APGIGYDEAVTLALGLHHSHRPRQERLARLPYYAAALPDVGLDGDPVGPPPAEDDGAAAEAYYG
  RIGNISVHIALNETRKIVNALLHRHGPILRLVMVETTRELKAGADERKRMIAEQAERERENAEI
  DVELRKSDRWMANARERRQRVRLARRQNNLCPYTSTPIGHADLLGDAYDIDHVIPLARGGRDSL
  DNMVLCQSDANKTKGDKTPWEAFHDKPGWIAQRDDFLARLDPQTAKALAWRFADDAGERVARKS
  AEDEDQGFLPRQLTDTGYIARVALRYLSLVTNEPNAVVATNGRLTGLLRLAWDITPGPAPRDLL
  PTPRDALRDDTAARRFLDGLTPPPLAKAVEGAVQARLAALGRSRVADAGLADALGLTLASLGGG
  GKNRADHRHHFIDAAMIAVTTRGLINQINQASGAGRILDLRKWPRTNFEPPYPTFRAEVMKQWD
  HIHPSIRPAHRDGGSLHAATVFGVRNRPDARVLVQRKPVEKLFLDANAKPLPADKIAEIIDGFA
  SPRMAKRFKALLARYQAAHPEVPPALAALAVARDPAFGPRGMTANTVIAGRSDGDGEDAGLITP
  FRANPKAAVRTMGNAVYEVWEIQVKGRPRWTHRVLTRFDRTQPAPPPPPENARLVMRLRRGDLV
  YWPLESGDRLFLVKKMAVDGRLALWPARLATGKATALYAQLSCPNINLNGDQGYCVQSAEGIRK
  EKIRTTSCTALGRLRLSKKAT

  SEQ ID NO: 367

  MKYTLGLDVGIASVGWAVIDKDNNKIIDLGVRCFDKAEESKTGESLATARRIARGMRRRISRRS
  QRLRLVKKLFVQYEIIKDSSEFNRIFDTSRDGWKDPWELRYNALSRILKPYELVQVLTHITKRR
  GFKSNRKEDLSTTKEGVVITSIKNNSEMLRTKNYRTIGEMIFMETPENSNKRNKVDEYIHTIAR
  EDLLNEIKYIFSIQRKLGSPFVTEKLEHDFLNIWEFQRPFASGDSILSKVGKCTLLKEELRAPT
  SCYTSEYFGLLQSINNLVLVEDNNTLTLNNDQRAKIIEYAHFKNEIKYSEIRKLLDIEPEILFK
  AHNLTHKNPSGNNESKKFYEMKSYHKLKSTLPTDIWGKLHSNKESLDNLFYCLTVYKNDNEIKD
  YLQANNLDYLIEYIAKLPTFNKFKHLSLVAMKRIIPFMEKGYKYSDACNMAELDFTGSSKLEKC
  NKLTVEPIIENVTNPVVIRALTQARKVINAIIQKYGLPYMVNIELAREAGMTRQDRDNLKKEHE
  NNRKAREKISDLIRQNGRVASGLDILKWRLWEDQGGRCAYSGKPIPVCDLLNDSLTQIDHIYPY
  SRSMDDSYMNKVLVLTDENQNKRSYTPYEVWGSTEKWEDFEARIYSMHLPQSKEKRLLNRNFIT
  KDLDSFISRNLNDTRYISRFLKNYIESYLQFSNDSPKSCVVCVNGQCTAQLRSRWGLNKNREES
  DLHHALDAAVIACADRKIIKEITNYYNERENHNYKVKYPLPWHSFRQDLMETLAGVFISRAPRR
  KITGPAHDETIRSPKHFNKGLTSVKIPLTTVTLEKLETMVKNTKGGISDKAVYNVLKNRLIEHN
  NKPLKAFAEKIYKPLKNGTNGAIIRSIRVETPSYTGVFRNEGKGISDNSLMVRVDVFKKKDKYY
  LVPIYVAHMIKKELPSKAIVPLKPESQWELIDSTHEFLFSLYQNDYLVIKTKKGITEGYYRSCH
  RGTGSLSLMPHFANNKNVKIDIGVRTAISIEKYNVDILGNKSIVKGEPRRGMEKYNSFKSN

  SEQ ID NO: 368

  MIRTLGIDIGIASIGWAVIEGEYTDKGLENKEIVASGVRVFTKAENPKNKESLALPRTLARSAR
  RRNARKKGRIQQVKHYLSKALGLDLECFVQGEKLATLFQTSKDFLSPWELRERALYRVLDKEEL
  ARVILHIAKRRGYDDITYGVEDNDSGKIKKAIAENSKRIKEEQCKTIGEMMYKLYFQKSLNVRN
  KKESYNRCVGRSELREELKTIFQIQQELKSPWVNEELIYKLLGNPDAQSKQEREGLIFYQRPLK
  GFGDKIGKCSHIKKGENSPYRACKHAPSAEEFVALTKSINFLKNLTNRHGLCFSQEDMCVYLGK
  ILQEAQKNEKGLTYSKLKLLLDLPSDFEFLGLDYSGKNPEKAVFLSLPSTFKLNKITQDRKTQD
  KIANILGANKDWEAILKELESLQLSKEQIQTIKDAKLNFSKHINLSLEALYHLLPLMREGKRYD
  EGVEILQERGIFSKPQPKNRQLLPPLSELAKEESYFDIPNPVLRRALSEFRKVVNALLEKYGGF
  HYFHIELTRDVCKAKSARMQLEKINKKNKSENDAASQLLEVLGLPNTYNNRLKCKLWKQQEEYC
  LYSGEKITIDHLKDQRALQIDHAFPLSRSLDDSQSNKVLCLTSSNQEKSNKTPYEWLGSDEKKW
  DMYVGRVYSSNFSPSKKRKLTQKNFKERNEEDFLARNLVDTGYIGRVTKEYIKHSLSFLPLPDG
  KKEHIRIISGSMTSTMRSFWGVQEKNRDHHLHHAQDAIIIACIEPSMIQKYTTYLKDKETHRLK
  SHQKAQILREGDHKLSLRWPMSNFKDKIQESIQNIIPSHHVSHKVTGELHQETVRTKEFYYQAF
  GGEEGVKKALKFGKIREINQGIVDNGAMVRVDIFKSKDKGKFYAVPIYTYDFAIGKLPNKAIVQ
  GKKNGIIKDWLEMDENYEFCFSLFKNDCIKIQTKEMQEAVLAIYKSTNSAKATIELEHLSKYAL
  KNEDEEKMFTDTDKEKNKTMTRESCGIQGLKVFQKVKLSVLGEVLEHKPRNRQNIALKTTPKHV

  SEQ ID NO: 369

  MKYSIGLDIGIASVGWSVINKDKERIEDMGVRIFQKAENPKDGSSLASSRREKRGSRRRNRRKK
  HRLDRIKNILCESGLVKKNEIEKIYKNAYLKSPWELRAKSLEAKISNKEIAQILLHIAKRRGFK
  SFRKTDRNADDTGKLLSGIQENKKIMEEKGYLTIGDMVAKDPKFNTHVRNKAGSYLFSFSRKLL
  EDEVRKIQAKQKELGNTHFTDDVLEKYIEVFNSQRNFDEGPSKPSPYYSEIGQIAKMIGNCTFE
  SSEKRTAKNTWSGERFVFLQKLNNFRIVGLSGKRPLTEEERDIVEKEVYLKKEVRYEKLRKILY
  LKEEERFGDLNYSKDEKQDKKTEKTKFISLIGNYTIKKLNLSEKLKSEIEEDKSKLDKIIEILT
  FNKSDKTIESNLKKLELSREDIEILLSEEFSGTLNLSLKAIKKILPYLEKGLSYNEACEKADYD
  YKNNGIKFKRGELLPVVDKDLIANPVVLRAISQTRKVVNAIIRKYGTPHTIHVEVARDLAKSYD
  DRQTIIKENKKRELENEKTKKFISEEFGIKNVKGKLLLKYRLYQEQEGRCAYSRKELSLSEVIL
  DESMTDIDHIIPYSRSMDDSYSNKVLVLSGENRKKSNLLPKEYFDRQGRDWDTFVLNVKAMKIH
  PRKKSNLLKEKFTREDNKDWKSRALNDTRYISRFVANYLENALEYRDDSPKKRVFMIPGQLTAQ
  LRARWRLNKVRENGDLHHALDAAVVAVTDQKAINNISNISRYKELKNCKDVIPSIEYHADEETG
  EVYFEEVKDTRFPMPWSGFDLELQKRLESENPREEFYNLLSDKRYLGWFNYEEGFIEKLRPVFV
  SRMPNRGVKGQAHQETIRSSKKISNQIAVSKKPLNSIKLKDLEKMQGRDTDRKLYEALKNRLEE
  YDDKPEKAFAEPFYKPTNSGKRGPLVRGIKVEEKQNVGVYVNGGQASNGSMVRIDVFRKNGKFY
  TVPIYVHQTLLKELPNRAINGKPYKDWDLIDGSFEFLYSFYPNDLIEIEFGKSKSIKNDNKLTK
  TEIPEVNLSEVLGYYRGMDTSTGAATIDTQDGKIQMRIGIKTVKNIKKYQVDVLGNVYKVKREK
  RQTF

  SEQ ID NO: 370

  MSKKVSRRYEEQAQEICQRLGSRPYSIGLDLGVGSIGVAVAAYDPIKKQPSDLVFVSSRIFIPS
  TGAAERRQKRGQRNSLRHRANRLKFLWKLLAERNLMLSYSEQDVPDPARLRFEDAVVRANPYEL
  RLKGLNEQLTLSELGYALYHIANHRGSSSVRTFLDEEKSSDDKKLEEQQAMTEQLAKEKGISTF
  IEVLTAFNTNGLIGYRNSESVKSKGVPVPTRDIISNEIDVLLQTQKQFYQEILSDEYCDRIVSA
  ILFENEKIVPEAGCCPYFPDEKKLPRCHFLNEERRLWEAINNARIKMPMQEGAAKRYQSASFSD
  EQRHILFHIARSGTDITPKLVQKEFPALKTSIIVLQGKEKAIQKIAGFRFRRLEEKSFWKRLSE
  EQKDDFFSAWTNTPDDKRLSKYLMKHLLLTENEVVDALKTVSLIGDYGPIGKTATQLLMKHLED
  GLTYTEALERGMETGEFQELSVWEQQSLLPYYGQILTGSTQALMGKYWHSAFKEKRDSEGFFKP
  NTNSDEEKYGRIANPVVHQTLNELRKLMNELITILGAKPQEITVELARELKVGAEKREDIIKQQ
  TKQEKEAVLAYSKYCEPNNLDKRYIERFRLLEDQAFVCPYCLEHISVADIAAGRADVDHIFPRD
  DTADNSYGNKVVAHRQCNDIKGKRTPYAAFSNTSAWGPIMHYLDETPGMWRKRRKFETNEEEYA
  KYLQSKGFVSRFESDNSYIAKAAKEYLRCLFNPNNVTAVGSLKGMETSILRKAWNLQGIDDLLG
  SRHWSKDADTSPTMRKNRDDNRHHGLDAIVALYCSRSLVQMINTMSEQGKRAVEIEAMIPIPGY
  ASEPNLSFEAQRELFRKKILEFMDLHAFVSMKTDNDANGALLKDTVYSILGADTQGEDLVFVVK
  KKIKDIGVKIGDYEEVASAIRGRITDKQPKWYPMEMKDKIEQLQSKNEAALQKYKESLVQAAAV
  LEESNRKLIESGKKPIQLSEKTISKKALELVGGYYYLISNNKRTKTFVVKEPSNEVKGFAFDTG
  SNLCLDFYHDAQGKLCGEIIRKIQAMNPSYKPAYMKQGYSLYVRLYQGDVCELRASDLTEAESN
  LAKTTHVRLPNAKPGRTFVIIITFTEMGSGYQIYFSNLAKSKKGQDTSFTLTTIKNYDVRKVQL
  SSAGLVRYVSPLLVDKIEKDEVALCGE

  SEQ ID NO: 371

  MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL
  ERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSND
  DVGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH
  QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKYAYSAD
  LFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRITKS
  GKPQFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDKEN
  IAQLTGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEFIL
  SPVVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIG
  KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKVL
  VKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQ
  KEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNHGYKHHA
  EDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQVDSEDNYSEMFIIPKQVQDIKDFRN
  FKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPEKFLMYQHD
  PRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQF
  KSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDKLKLGKAIDKNAK
  FIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEPRIKKTIGKKVN
  SIEKLTTDVLGNVFTNTQYTKPQLLFKRGN

  SEQ ID NO: 372

  MIMKLEKWRLGLDLGTNSIGWSVFSLDKDNSVQDLIDMGVRIFSDGRDPKTKEPLAVARRTARS
  QRKLIYRRKLRRKQVFKFLQEQGLFPKTKEECMTLKSLNPYELRIKALDEKLEPYELGRALFNL
  AVRRGFKSNRKDGSREEVSEKKSPDEIKTQADMQTHLEKAIKENGCRTITEFLYKNQGENGGIR
  FAPGRMTYYPTRKMYEEEFNLIRSKQEKYYPQVDWDDIYKAIFYQRPLKPQQRGYCIYENDKER
  TFKAMPCSQKLRILQDIGNLAYYEGGSKKRVELNDNQDKVLYELLNSKDKVTFDQMRKALCLAD
  SNSFNLEENRDFLIGNPTAVKMRSKNRFGKLWDEIPLEEQDLIIETIITADEDDAVYEVIKKYD
  LTQEQRDFIVKNTILQSGTSMLCKEVSEKLVKRLEEIADLKYHEAVESLGYKFADQTVEKYDLL
  PYYGKVLPGSTMEIDLSAPETNPEKHYGKISNPTVHVALNQTRVVVNALIKEYGKPSQIAIELS
  RDLKNNVEKKAEIARKQNQRAKENIAINDTISALYHTAFPGKSFYPNRNDRMKYRLWSELGLGN
  KCIYCGKGISGAELFTKEIEIEHILPFSRTLLDAESNLTVAHSSCNAFKAERSPFEAFGTNPSG
  YSWQEIIQRANQLKNTSKKNKFSPNAMDSFEKDSSFIARQLSDNQYIAKAALRYLKCLVENPSD
  VWTTNGSMTKLLRDKWEMDSILCRKFTEKEVALLGLKPEQIGNYKKNRFDHRHHAIDAVVIGLT
  DRSMVQKLATKNSHKGNRIEIPEFPILRSDLIEKVKNIVVSFKPDHGAEGKLSKETLLGKIKLH
  GKETFVCRENIVSLSEKNLDDIVDEIKSKVKDYVAKHKGQKIEAVLSDFSKENGIKKVRCVNRV
  QTPIEITSGKISRYLSPEDYFAAVIWEIPGEKKTFKAQYIRRNEVEKNSKGLNVVKPAVLENGK
  PHPAAKQVCLLHKDDYLEFSDKGKMYFCRIAGYAATNNKLDIRPVYAVSYCADWINSTNETMLT
  GYWKPTPTQNWVSVNVLFDKQKARLVTVSPIGRVFRK

  SEQ ID NO: 373

  MSSKAIDSLEQLDLFKPQEYTLGLDLGIKSIGWAILSGERIANAGVYLFETAEELNSTGNKLIS
  KAAERGRKRRIRRMLDRKARRGRHIRYLLEREGLPTDELEEVVVHQSNRTLWDVRAEAVERKLT
  KQELAAVLFHLVRHRGYFPNTKKLPPDDESDSADEEQGKINRATSRLREELKASDCKTIGQFLA
  QNRDRQRNREGDYSNLMARKLVFEEALQILAFQRKQGHELSKDFEKTYLDVLMGQRSGRSPKLG
  NCSLIPSELRAPSSAPSTEWFKFLQNLGNLQISNAYREEWSIDAPRRAQIIDACSQRSTSSYWQ
  IRRDFQIPDEYRFNLVNYERRDPDVDLQEYLQQQERKTLANFRNWKQLEKIIGTGHPIQTLDEA
  ARLITLIKDDEKLSDQLADLLPEASDKAITQLCELDFTTAAKISLEAMYRILPHMNQGMGFFDA
  CQQESLPEIGVPPAGDRVPPFDEMYNPVVNRVLSQSRKLINAVIDEYGMPAKIRVELARDLGKG
  RELRERIKLDQLDKSKQNDQRAEDFRAEFQQAPRGDQSLRYRLWKEQNCTCPYSGRMIPVNSVL
  SEDTQIDHILPISQSFDNSLSNKVLCFTEENAQKSNRTPFEYLDAADFQRLEAISGNWPEAKRN
  KLLHKSFGKVAEEWKSRALNDTRYLTSALADHLRHHLPDSKIQTVNGRITGYLRKQWGLEKDRD
  KHTHHAVDAIVVACTTPAIVQQVTLYHQDIRRYKKLGEKRPTPWPETFRQDVLDVEEEIFITRQ
  PKKVSGGIQTKDTLRKHRSKPDRQRVALTKVKLADLERLVEKDASNRNLYEHLKQCLEESGDQP
  TKAFKAPFYMPSGPEAKQRPILSKVTLLREKPEPPKQLTELSGGRRYDSMAQGRLDIYRYKPGG
  KRKDEYRVVLQRMIDLMRGEENVHVFQKGVPYDQGPEIEQNYTFLFSLYFDDLVEFQRSADSEV
  IRGYYRTFNIANGQLKISTYLEGRQDFDFFGANRLAHFAKVQVNLLGKVIK

  SEQ ID NO: 374

  MRSLRYRLALDLGSTSLGWALFRLDACNRPTAVIKAGVRIFSDGRNPKDGSSLAVTRRAARAMR
  RRRDRLLKRKTRMQAKLVEHGFFPADAGKRKALEQLNPYALRAKGLQEALLPGEFARALFHINQ
  RRGFKSNRKTDKKDNDSGVLKKAIGQLRQQMAEQGSRTVGEYLWTRLQQGQGVRARYREKPYTT
  EEGKKRIDKSYDLYIDRAMIEQEFDALWAAQAAFNPTLFHEAARADLKDTLLHQRPLRPVKPGR
  CTLLPEEERAPLALPSTQRFRIHQEVNHLRLLDENLREVALTLAQRDAVVTALETKAKLSFEQI
  RKLLKLSGSVQFNLEDAKRTELKGNATSAALARKELFGAAWSGFDEALQDEIVWQLVTEEGEGA
  LIAWLQTHTGVDEARAQAIVDVSLPEGYGNLSRKALARIVPALRAAVITYDKAVQAAGFDHHSQ
  LGFEYDASEVEDLVHPETGEIRSVFKQLPYYGKALQRHVAFGSGKPEDPDEKRYGKIANPTVHI
  GLNQVRMVVNALIRRYGRPTEVVIELARDLKQSREQKVEAQRRQADNQRRNARIRRSIAEVLGI
  GEERVRGSDIQKWICWEELSFDAADRRCPYSGVQISAAMLLSDEVEVEHILPFSKTLDDSLNNR
  TVAMRQANRIKRNRTPWDARAEFEAQGWSYEDILQRAERMPLRKRYRFAPDGYERWLGDDKDFL
  ARALNDTRYLSRVAAEYLRLVCPGTRVIPGQLTALLRGKFGLNDVLGLDGEKNRNDHRHHAVDA
  CVIGVTDQGLMQRFATASAQARGDGLTRLVDGMPMPWPTYRDHVERAVRHIWVSHRPDHGFEGA
  MMEETSYGIRKDGSIKQRRKADGSAGREISNLIRIHEATQPLRHGVSADGQPLAYKGYVGGSNY
  CIEITVNDKGKWEGEVISTFRAYGVVRAGGMGRLRNPHEGQNGRKLIMRLVIGDSVRLEVDGAE
  RTMRIVKISGSNGQIFMAPIHEANVDARNTDKQDAFTYTSKYAGSLQKAKTRRVTISPIGEVRD
  PGFKG

  SEQ ID NO: 375

  MARPAFRAPRREHVNGWTPDPHRISKPFFILVSWHLLSRVVIDSSSGCFPGTSRDHTDKFAEWE
  CAVQPYRLSFDLGTNSIGWGLLNLDRQGKPREIRALGSRIFSDGRDPQDKASLAVARRLARQMR
  RRRDRYLTRRTRLMGALVRFGLMPADPAARKRLEVAVDPYLARERATRERLEPFEIGRALFHLN
  QRRGYKPVRTATKPDEEAGKVKEAVERLEAAIAAAGAPTLGAWFAWRKTRGETLRARLAGKGKE
  AAYPFYPARRMLEAEFDTLWAEQARHHPDLLTAEAREILRHRIFHQRPLKPPPVGRCTLYPDDG
  RAPRALPSAQRLRLFQELASLRVIHLDLSERPLTPAERDRIVAFVQGRPPKAGRKPGKVQKSVP
  FEKLRGLLELPPGTGFSLESDKRPELLGDETGARIAPAFGPGWTALPLEEQDALVELLLTEAEP
  ERAIAALTARWALDEATAAKLAGATLPDFHGRYGRRAVAELLPVLERETRGDPDGRVRPIRLDE
  AVKLLRGGKDHSDFSREGALLDALPYYGAVLERHVAFGTGNPADPEEKRVGRVANPTVHIALNQ
  LRHLVNAILARHGRPEEIVIELARDLKRSAEDRRREDKRQADNQKRNEERKRLILSLGERPTPR
  NLLKLRLWEEQGPVENRRCPYSGETISMRMLLSEQVDIDHILPFSVSLDDSAANKVVCLREANR
  IKRNRSPWEAFGHDSERWAGILARAEALPKNKRWRFAPDALEKLEGEGGLRARHLNDTRHLSRL
  AVEYLRCVCPKVRVSPGRLTALLRRRWGIDAILAEADGPPPEVPAETLDPSPAEKNRADHRHHA
  LDAVVIGCIDRSMVQRVQLAAASAEREAAAREDNIRRVLEGFKEEPWDGFRAELERRARTIVVS
  HRPEHGIGGALHKETAYGPVDPPEEGFNLVVRKPIDGLSKDEINSVRDPRLRRALIDRLAIRRR
  DANDPATALAKAAEDLAAQPASRGIRRVRVLKKESNPIRVEHGGNPSGPRSGGPFHKLLLAGEV
  HHVDVALRADGRRWVGHWVTLFEAHGGRGADGAAAPPRLGDGERFLMRLHKGDCLKLEHKGRVR
  VMQVVKLEPSSNSVVVVEPHQVKTDRSKHVKISCDQLRARGARRVTVDPLGRVRVHAPGARVGI
  GGDAGRTAMEPAEDIS

  SEQ ID NO: 376

  MKRTSLRAYRLGVDLGANSLGWFVVWLDDHGQPEGLGPGGVRIFPDGRNPQSKQSNAAGRRLAR
  SARRRRDRYLQRRGKLMGLLVKHGLMPADEPARKRLECLDPYGLRAKALDEVLPLHHVGRALFH
  LNQRRGLFANRAIEQGDKDASAIKAAAGRLQTSMQACGARTLGEFLNRRHQLRATVRARSPVGG
  DVQARYEFYPTRAMVDAEFEAIWAAQAPHHPTMTAEAHDTIREAIFSQRAMKRPSIGKCSLDPA
  TSQDDVDGFRCAWSHPLAQRFRIWQDVRNLAVVETGPTSSRLGKEDQDKVARALLQTDQLSFDE
  IRGLLGLPSDARFNLESDRRDHLKGDATGAILSARRHFGPAWHDRSLDRQIDIVALLESALDEA
  AIIASLGTTHSLDEAAAQRALSALLPDGYCRLGLRAIKRVLPLMEAGRTYAEAASAAGYDHALL
  PGGKLSPTGYLPYYGQWLQNDVVGSDDERDTNERRWGRLPNPTVHIGIGQLRRVVNELIRWHGP
  PAEITVELTRDLKLSPRRLAELEREQAENQRKNDKRTSLLRKLGLPASTHNLLKLRLWDEQGDV
  ASECPYTGEAIGLERLVSDDVDIDHLIPFSISWDDSAANKVVCMRYANREKGNRTPFEAFGHRQ
  GRPYDWADIAERAARLPRGKRWRFGPGARAQFEELGDFQARLLNETSWLARVAKQYLAAVTHPH
  RIHVLPGRLTALLRATWELNDLLPGSDDRAAKSRKDHRHHAIDALVAALTDQALLRRMANAHDD
  TRRKIEVLLPWPTFRIDLETRLKAMLVSHKPDHGLQARLHEDTAYGTVEHPETEDGANLVYRKT
  FVDISEKEIDRIRDRRLRDLVRAHVAGERQQGKTLKAAVLSFAQRRDIAGHPNGIRHVRLTKSI
  KPDYLVPIRDKAGRIYKSYNAGENAFVDILQAESGRWIARATTVFQANQANESHDAPAAQPIMR
  VFKGDMLRIDHAGAEKFVKIVRLSPSNNLLYLVEHHQAGVFQTRHDDPEDSFRWLFASFDKLRE
  WNAELVRIDTLGQPWRRKRGLETGSEDATRIGWTRPKKWP

  SEQ ID NO: 377

  MERIFGFDIGTTSIGFSVIDYSSTQSAGNIQRLGVRIFPEARDPDGTPLNQQRRQKRMMRRQLR
  RRRIRRKALNETLHEAGFLPAYGSADWPVVMADEPYELRRRGLEEGLSAYEFGRAIYHLAQHRH
  FKGRELEESDTPDPDVDDEKEAANERAATLKALKNEQTTLGAWLARRPPSDRKRGIHAHRNVVA
  EEFERLWEVQSKFHPALKSEEMRARISDTIFAQRPVFWRKNTLGECRFMPGEPLCPKGSWLSQQ
  RRMLEKLNNLAIAGGNARPLDAEERDAILSKLQQQASMSWPGVRSALKALYKQRGEPGAEKSLK
  FNLELGGESKLLGNALEAKLADMFGPDWPAHPRKQEIRHAVHERLWAADYGETPDKKRVIILSE
  KDRKAHREAAANSFVADFGITGEQAAQLQALKLPTGWEPYSIPALNLFLAELEKGERFGALVNG
  PDWEGWRRTNFPHRNQPTGEILDKLPSPASKEERERISQLRNPTVVRTQNELRKVVNNLIGLYG
  KPDRIRIEVGRDVGKSKREREEIQSGIRRNEKQRKKATEDLIKNGIANPSRDDVEKWILWKEGQ
  ERCPYTGDQIGFNALFREGRYEVEHIWPRSRSFDNSPRNKTLCRKDVNIEKGNRMPFEAFGHDE
  DRWSAIQIRLQGMVSAKGGTGMSPGKVKRFLAKTMPEDFAARQLNDTRYAAKQILAQLKRLWPD
  MGPEAPVKVEAVTGQVTAQLRKLWTLNNILADDGEKTRADHRHHAIDALTVACTHPGMTNKLSR
  YWQLRDDPRAEKPALTPPWDTIRADAEKAVSEIVVSHRVRKKVSGPLHKETTYGDTGTDIKTKS
  GTYRQFVTRKKIESLSKGELDEIRDPRIKEIVAAHVAGRGGDPKKAFPPYPCVSPGGPEIRKVR
  LTSKQQLNLMAQTGNGYADLGSNHHIAIYRLPDGKADFEIVSLFDASRRLAQRNPIVQRTRADG
  ASFVMSLAAGEAIMIPEGSKKGIWIVQGVWASGQVVLERDTDADHSTTTRPMPNPILKDDAKKV
  SIDPIGRVRPSND

  SEQ ID NO: 378

  MNKRILGLDTGTNSLGWAVVDWDEHAQSYELIKYGDVIFQEGVKIEKGIESSKAAERSGYKAIR
  KQYFRRRLRKIQVLKVLVKYHLCPYLSDDDLRQWHLQKQYPKSDELMLWQRTSDEEGKNPYYDR
  HRCLHEKLDLTVEADRYTLGRALYHLTQRRGFLSNRLDTSADNKEDGVVKSGISQLSTEMEEAG
  CEYLGDYFYKLYDAQGNKVRIRQRYTDRNKHYQHEFDAICEKQELSSELIEDLQRAIFFQLPLK
  SQRHGVGRCTFERGKPRCADSHPDYEEFRMLCFVNNIQVKGPHDLELRPLTYEEREKIEPLFFR
  KSKPNFDFEDIAKALAGKKNYAWIHDKEERAYKFNYRMTQGVPGCPTIAQLKSIFGDDWKTGIA
  ETYTLIQKKNGSKSLQEMVDDVWNVLYSFSSVEKLKEFAHHKLQLDEESAEKFAKIKLSHSFAA
  LSLKAIRKFLPFLRKGMYYTHASFFANIPTIVGKEIWNKEQNRKYIMENVGELVFNYQPKHREV
  QGTIEMLIKDFLANNFELPAGATDKLYHPSMIETYPNAQRNEFGILQLGSPRTNAIRNPMAMRS
  LHILRRVVNQLLKESIIDENTEVHVEYARELNDANKRRAIADRQKEQDKQHKKYGDEIRKLYKE
  ETGKDIEPTQTDVLKFQLWEEQNHHCLYTGEQIGITDFIGSNPKFDIEHTIPQSVGGDSTQMNL
  TLCDNRFNREVKKAKLPTELANHEEILTRIEPWKNKYEQLVKERDKQRTFAGMDKAVKDIRIQK
  RHKLQMEIDYWRGKYERFTMTEVPEGFSRRQGTGIGLISRYAGLYLKSLFHQADSRNKSNVYVV
  KGVATAEFRKMWGLQSEYEKKCRDNHSHHCMDAITIACIGKREYDLMAEYYRMEETFKQGRGSK
  PKFSKPWATFTEDVLNIYKNLLVVHDTPNNMPKHTKKYVQTSIGKVLAQGDTARGSLHLDTYYG
  AIERDGEIRYVVRRPLSSFTKPEELENIVDETVKRTIKEAIADKNFKQAIAEPIYMNEEKGILI
  KKVRCFAKSVKQPINIRQHRDLSKKEYKQQYHVMNENNYLLAIYEGLVKNKVVREFEIVSYIEA
  AKYYKRSQDRNIFSSIVPTHSTKYGLPLKTKLLMGQLVLMFEENPDEIQVDNTKDLVKRLYKVV
  GIEKDGRIKFKYHQEARKEGLPIFSTPYKNNDDYAPIFRQSINNINILVDGIDFTIDILGKVTL
  KE

  SEQ ID NO: 379

  MNYKMGLDIGIASVGWAVINLDLKRIEDLGVRIFDKAEHPQNGESLALPRRIARSARRRLRRRK
  HRLERIRRLLVSENVLTKEEMNLLFKQKKQIDVWQLRVDALERKLNNDELARVLLHLAKRRGFK
  SNRKSERNSKESSEFLKNIEENQSILAQYRSVGEMIVKDSKFAYHKRNKLDSYSNMIARDDLER
  EIKLIFEKQREFNNPVCTERLEEKYLNIWSSQRPFASKEDIEKKVGFCTFEPKEKRAPKATYTF
  QSFIVWEHINKLRLVSPDETRALTEIERNLLYKQAFSKNKMTYYDIRKLLNLSDDIHFKGLLYD
  PKSSLKQIENIRFLELDSYHKIRKCIENVYGKDGIRMFNETDIDTFGYALTIFKDDEDIVAYLQ
  NEYITKNGKRVSNLANKVYDKSLIDELLNLSFSKFAHLSMKAIRNILPYMEQGEIYSKACELAG
  YNFTGPKKKEKALLLPVIPNIANPVVMRALTQSRKVVNAIIKKYGSPVSIHIELARDLSHSFDE
  RKKIQKDQTENRKKNETAIKQLIEYELTKNPTGLDIVKFKLWSEQQGRCMYSLKPIELERLLEP
  GYVEVDHILPYSRSLDDSYANKVLVLTKENREKGNHTPVEYLGLGSERWKKFEKFVLANKQFSK
  KKKQNLLRLRYEETEEKEFKERNLNDTRYISKFFANFIKEHLKFADGDGGQKVYTINGKITAHL
  RSRWDFNKNREESDLHHAVDAVIVACATQGMIKKITEFYKAREQNKESAKKKEPIFPQPWPHFA
  DELKARLSKFPQESIEAFALGNYDRKKLESLRPVFVSRMPKRSVTGAAHQETLRRCVGIDEQSG
  KIQTAVKTKLSDIKLDKDGHFPMYQKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEP
  GPVIRTVKIIDTKNKVVHLDGSKTVAYNSNIVRTDVFEKDGKYYCVPVYTMDIMKGTLPNKAIE
  ANKPYSEWKEMTEEYTFQFSLFPNDLVRIVLPREKTIKTSTNEEIIIKDIFAYYKTIDSATGGL
  ELISHDRNFSLRGVGSKTLKRFEKYQVDVLGNIHKVKGEKRVGLAAPTNQKKGKTVDSLQSVSD

  SEQ ID NO: 380

  MRRLGLDLGTNSIGWCLLDLGDDGEPVSIFRTGARIFSDGRDPKSLGSLKATRREARLTRRRRD
  RFIQRQKNLINALVKYGLMPADEIQRQALAYKDPYPIRKKALDEAIDPYEMGRAIFHINQRRGF
  KSNRKSADNEAGVVKQSIADLEMKLGEAGARTIGEFLADRQATNDTVRARRLSGTNALYEFYPD
  RYMLEQEFDTLWAKQAAFNPSLYIEAARERLKEIVFFQRKLKPQEVGRCIFLSDEDRISKALPS
  FQRFRIYQELSNLAWIDHDGVAHRITASLALRDHLFDELEHKKKLTFKAMRAILRKQGVVDYPV
  GFNLESDNRDHLIGNLTSCIMRDAKKMIGSAWDRLDEEEQDSFILMLQDDQKGDDEVRSILTQQ
  YGLSDDVAEDCLDVRLPDGHGSLSKKAIDRILPVLRDQGLIYYDAVKEAGLGEANLYDPYAALS
  DKLDYYGKALAGHVMGASGKFEDSDEKRYGTISNPTVHIALNQVRAVVNELIRLHGKPDEVVIE
  IGRDLPMGADGKRELERFQKEGRAKNERARDELKKLGHIDSRESRQKFQLWEQLAKEPVDRCCP
  FTGKMMSISDLFSDKVEIEHLLPFSLTLDDSMANKTVCFRQANRDKGNRAPFDAFGNSPAGYDW
  QEILGRSQNLPYAKRWRFLPDAMKRFEADGGFLERQLNDTRYISRYTTEYISTIIPKNKIWVVT
  GRLTSLLRGFWGLNSILRGHNTDDGTPAKKSRDDHRHHAIDAIVVGMTSRGLLQKVSKAARRSE
  DLDLTRLFEGRIDPWDGFRDEVKKHIDAIIVSHRPRKKSQGALHNDTAYGIVEHAENGASTVVH
  RVPITSLGKQSDIEKVRDPLIKSALLNETAGLSGKSFENAVQKWCADNSIKSLRIVETVSIIPI
  TDKEGVAYKGYKGDGNAYMDIYQDPTSSKWKGEIVSRFDANQKGFIPSWQSQFPTARLIMRLRI
  NDLLKLQDGEIEEIYRVQRLSGSKILMAPHTEANVDARDRDKNDTFKLTSKSPGKLQSASARKV
  HISPTGLIREG

  SEQ ID NO: 381

  MKNILGLDLGLSSIGWSVIRENSEEQELVAMGSRVVSLTAAELSSFTQGNGVSINSQRTQKRTQ
  RKGYDRYQLRRTLLRNKLDTLGMLPDDSLSYLPKLQLWGLRAKAVTQRIELNELGRVLLHLNQK
  RGYKSIKSDFSGDKKITDYVKTVKTRYDELKEMRLTIGELFFRRLTENAFFRCKEQVYPRQAYV
  EEFDCIMNCQRKFYPDILTDETIRCIRDEIIYYQRPLKSCKYLVSRCEFEKRFYLNAAGKKTEA
  GPKVSPRTSPLFQVCRLWESINNIVVKDRRNEIVFISAEQRAALFDFLNTHEKLKGSDLLKLLG
  LSKTYGYRLGEQFKTGIQGNKTRVEIERALGNYPDKKRLLQFNLQEESSSMVNTETGEIIPMIS
  LSFEQEPLYRLWHVLYSIDDREQLQSVLRQKFGIDDDEVLERLSAIDLVKAGFGNKSSKAIRRI
  LPFLQLGMNYAEACEAAGYNHSNNYTKAENEARALLDRLPAIKKNELRQPVVEKILNQMVNVVN
  ALMEKYGRFDEIRVELARELKQSKEERSNTYKSINKNQRENEQIAKRIVEYGVPTRSRIQKYKM
  WEESKHCCIYCGQPVDVGDFLRGFDVEVEHIIPKSLYFDDSFANKVCSCRSCNKEKNNRTAYDY
  MKSKGEKALSDYVERVNTMYTNNQISKTKWQNLLTPVDKISIDFIDRQLRESQYIARKAKEILT
  SICYNVTATSGSVTSFLRHVWGWDTVLHDLNFDRYKKVGLTEVIEVNHRGSVIRREQIKDWSKR
  FDHRHHAIDALTIACTKQAYIQRLNNLRAEEGPDFNKMSLERYIQSQPHFSVAQVREAVDRILV
  SFRAGKRAVTPGKRYIRKNRKRISVQSVLIPRGALSEESVYGVIHVWEKDEQGHVIQKQRAVMK
  YPITSINREMLDKEKVVDKRIHRILSGRLAQYNDNPKEAFAKPVYIDKECRIPIRTVRCFAKPA
  INTLVPLKKDDKGNPVAWVNPGNNHHVAIYRDEDGKYKERTVTFWEAVDRCRVGIPAIVTQPDT
  IWDNILQRNDISENVLESLPDVKWQFVLSLQQNEMFILGMNEEDYRYAMDQQDYALLNKYLYRV
  QKLSKSDYSFRYHTETSVEDKYDGKPNLKLSMQMGKLKRVSIKSLLGLNPHKVHISVLGEIKEI
  S

  SEQ ID NO: 382

  MAEKQHRWGLDIGTNSIGWAVIALIEGRPAGLVATGSRIFSDGRNPKDGSSLAVERRGPRQMRR
  RRDRYLRRRDRFMQALINVGLMPGDAAARKALVTENPYVLRQRGLDQALTLPEFGRALFHLNQR
  RGFQSNRKTDRATAKESGKVKNAIAAFRAGMGNARTVGEALARRLEDGRPVRARMVGQGKDEHY
  ELYIAREWIAQEFDALWASQQRFHAEVLADAARDRLRAILLFQRKLLPVPVGKCFLEPNQPRVA
  AALPSAQRFRLMQELNHLRVMTLADKRERPLSFQERNDLLAQLVARPKCGFDMLRKIVFGANKE
  AYRFTIESERRKELKGCDTAAKLAKVNALGTRWQALSLDEQDRLVCLLLDGENDAVLADALREH
  YGLTDAQIDTLLGLSFEDGHMRLGRSALLRVLDALESGRDEQGLPLSYDKAVVAAGYPAHTADL
  ENGERDALPYYGELLWRYTQDAPTAKNDAERKFGKIANPTVHIGLNQLRKLVNALIQRYGKPAQ
  IVVELARNLKAGLEEKERIKKQQTANLERNERIRQKLQDAGVPDNRENRLRMRLFEELGQGNGL
  GTPCIYSGRQISLQRLFSNDVQVDHILPFSKTLDDSFANKVLAQHDANRYKGNRGPFEAFGANR
  DGYAWDDIRARAAVLPRNKRNRFAETAMQDWLHNETDFLARQLTDTAYLSRVARQYLTAICSKD
  DVYVSPGRLTAMLRAKWGLNRVLDGVMEEQGRPAVKNRDDHRHHAIDAVVIGATDRAMLQQVAT
  LAARAREQDAERLIGDMPTPWPNFLEDVRAAVARCVVSHKPDHGPEGGLHNDTAYGIVAGPFED
  GRYRVRHRVSLFDLKPGDLSNVRCDAPLQAELEPIFEQDDARAREVALTALAERYRQRKVWLEE
  LMSVLPIRPRGEDGKTLPDSAPYKAYKGDSNYCYELFINERGRWDGELISTFRANQAAYRRFRN
  DPARFRRYTAGGRPLLMRLCINDYIAVGTAAERTIFRVVKMSENKITLAEHFEGGTLKQRDADK
  DDPFKYLTKSPGALRDLGARRIFVDLIGRVLDPGIKGD

  SEQ ID NO: 383

  MIERILGVDLGISSLGWAIVEYDKDDEAANRIIDCGVRLFTAAETPKKKESPNKARREARGIRR
  VLNRRRVRMNMIKKLFLRAGLIQDVDLDGEGGMFYSKANRADVWELRHDGLYRLLKGDELARVL
  IHIAKHRGYKFIGDDEADEESGKVKKAGVVLRQNFEAAGCRTVGEWLWRERGANGKKRNKHGDY
  EISIHRDLLVEEVEAIFVAQQEMRSTIATDALKAAYREIAFFVRPMQRIEKMVGHCTYFPEERR
  APKSAPTAEKFIAISKFFSTVIIDNEGWEQKIIERKTLEELLDFAVSREKVEFRHLRKFLDLSD
  NEIFKGLHYKGKPKTAKKREATLFDPNEPTELEFDKVEAEKKAWISLRGAAKLREALGNEFYGR
  FVALGKHADEATKILTYYKDEGQKRRELTKLPLEAEMVERLVKIGFSDFLKLSLKAIRDILPAM
  ESGARYDEAVLMLGVPHKEKSAILPPLNKTDIDILNPTVIRAFAQFRKVANALVRKYGAFDRVH
  FELAREINTKGEIEDIKESQRKNEKERKEAADWIAETSFQVPLTRKNILKKRLYIQQDGRCAYT
  GDVIELERLFDEGYCEIDHILPRSRSADDSFANKVLCLARANQQKTDRTPYEWFGHDAARWNAF
  ETRTSAPSNRVRTGKGKIDRLLKKNFDENSEMAFKDRNLNDTRYMARAIKTYCEQYWVFKNSHT
  KAPVQVRSGKLTSVLRYQWGLESKDRESHTHHAVDAIIIAFSTQGMVQKLSEYYRFKETHREKE
  RPKLAVPLANFRDAVEEATRIENTETVKEGVEVKRLLISRPPRARVTGQAHEQTAKPYPRIKQV
  KNKKKWRLAPIDEEKFESFKADRVASANQKNFYETSTIPRVDVYHKKGKFHLVPIYLHEMVLNE
  LPNLSLGTNPEAMDENFFKFSIFKDDLISIQTQGTPKKPAKIIMGYFKNMHGANMVLSSINNSP
  CEGFTCTPVSMDKKHKDKCKLCPEENRIAGRCLQGFLDYWSQEGLRPPRKEFECDQGVKFALDV
  KKYQIDPLGYYYEVKQEKRLGTIPQMRSAKKLVKK

  SEQ ID NO: 384

  MNNSIKSKPEVTIGLDLGVGSVGWAIVDNETNIIHHLGSRLFSQAKTAEDRRSFRGVRRLIRRR
  KYKLKRFVNLIWKYNSYFGFKNKEDILNNYQEQQKLHNTVLNLKSEALNAKIDPKALSWILHDY
  LKNRGHFYEDNRDFNVYPTKELAKYFDKYGYYKGIIDSKEDNDNKLEEELTKYKFSNKHWLEEV
  KKVLSNQTGLPEKFKEEYESLFSYVRNYSEGPGSINSVSPYGIYHLDEKEGKVVQKYNNIWDKT
  IGKCNIFPDEYRAPKNSPIAMIFNEINELSTIRSYSIYLTGWFINQEFKKAYLNKLLDLLIKTN
  GEKPIDARQFKKLREETIAESIGKETLKDVENEEKLEKEDHKWKLKGLKLNTNGKIQYNDLSSL
  AKFVHKLKQHLKLDFLLEDQYATLDKINFLQSLFVYLGKHLRYSNRVDSANLKEFSDSNKLFER
  ILQKQKDGLFKLFEQTDKDDEKILAQTHSLSTKAMLLAITRMTNLDNDEDNQKNNDKGWNFEAI
  KNFDQKFIDITKKNNNLSLKQNKRYLDDRFINDAILSPGVKRILREATKVFNAILKQFSEEYDV
  TKVVIELARELSEEKELENTKNYKKLIKKNGDKISEGLKALGISEDEIKDILKSPTKSYKFLLW
  LQQDHIDPYSLKEIAFDDIFTKTEKFEIDHIIPYSISFDDSSSNKLLVLAESNQAKSNQTPYEF
  ISSGNAGIKWEDYEAYCRKFKDGDSSLLDSTQRSKKFAKMMKTDTSSKYDIGFLARNLNDTRYA
  TIVFRDALEDYANNHLVEDKPMFKVVCINGSVTSFLRKNFDDSSYAKKDRDKNIHHAVDASIIS
  IFSNETKTLFNQLTQFADYKLFKNTDGSWKKIDPKTGVVTEVTDENWKQIRVRNQVSEIAKVIE
  KYIQDSNIERKARYSRKIENKTNISLFNDTVYSAKKVGYEDQIKRKNLKTLDIHESAKENKNSK
  VKRQFVYRKLVNVSLLNNDKLADLFAEKEDILMYRANPWVINLAEQIFNEYTENKKIKSQNVFE
  KYMLDLTKEFPEKFSEFLVKSMLRNKTAIIYDDKKNIVHRIKRLKMLSSELKENKLSNVIIRSK
  NQSGTKLSYQDTINSLALMIMRSIDPTAKKQYIRVPLNTLNLHLGDHDFDLHNMDAYLKKPKFV
  KYLKANEIGDEYKPWRVLTSGTLLIHKKDKKLMYISSFQNLNDVIEIKNLIETEYKENDDSDSK
  KKKKANRFLMTLSTILNDYILLDAKDNFDILGLSKNRIDEILNSKLGLDKIVK

  SEQ ID NO: 385

  MGGSEVGTVPVTWRLGVDVGERSIGLAAVSYEEDKPKEILAAVSWIHDGGVGDERSGASRLALR
  GMARRARRLRRFRRARLRDLDMLLSELGWTPLPDKNVSPVDAWLARKRLAEEYVVDETERRRLL
  GYAVSHMARHRGWRNPWTTIKDLKNLPQPSDSWERTRESLEARYSVSLEPGTVGQWAGYLLQRA
  PGIRLNPTQQSAGRRAELSNATAFETRLRQEDVLWELRCIADVQGLPEDVVSNVIDAVFCQKRP
  SVPAERIGRDPLDPSQLRASRACLEFQEYRIVAAVANLRIRDGSGSRPLSLEERNAVIEALLAQ
  TERSLTWSDIALEILKLPNESDLTSVPEEDGPSSLAYSQFAPFDETSARIAEFIAKNRRKIPTF
  AQWWQEQDRTSRSDLVAALADNSIAGEEEQELLVHLPDAELEALEGLALPSGRVAYSRLTLSGL
  TRVMRDDGVDVHNARKTCFGVDDNWRPPLPALHEATGHPVVDRNLAILRKFLSSATMRWGPPQS
  IVVELARGASESRERQAEEEAARRAHRKANDRIRAELRASGLSDPSPADLVRARLLELYDCHCM
  YCGAPISWENSELDHIVPRTDGGSNRHENLAITCGACNKEKGRRPFASWAETSNRVQLRDVIDR
  VQKLKYSGNMYWTRDEFSRYKKSVVARLKRRTSDPEVIQSIESTGYAAVALRDRLLSYGEKNGV
  AQVAVFRGGVTAEARRWLDISIERLFSRVAIFAQSTSTKRLDRRHHAVDAVVLTTLTPGVAKTL
  ADARSRRVSAEFWRRPSDVNRHSTEEPQSPAYRQWKESCSGLGDLLISTAARDSIAVAAPLRLR
  PTGALHEETLRAFSEHTVGAAWKGAELRRIVEPEVYAAFLALTDPGGRFLKVSPSEDVLPADEN
  RHIVLSDRVLGPRDRVKLFPDDRGSIRVRGGAAYIASFHHARVFRWGSSHSPSFALLRVSLADL
  AVAGLLRDGVDVFTAELPPWTPAWRYASIALVKAVESGDAKQVGWLVPGDELDFGPEGVTTAAG
  DLSMFLKYFPERHWVVTGFEDDKRINLKPAFLSAEQAEVLRTERSDRPDTLTEAGEILAQFFPR
  CWRATVAKVLCHPGLTVIRRTALGQPRWRRGHLPYSWRPWSADPWSGGTP

  SEQ ID NO: 386

  MHNKKNITIGFDLGIASIGWAIIDSTTSKILDWGTRTFEERKTANERRAFRSTRRNIRRKAYRN
  QRFINLILKYKDLFELKNISDIQRANKKDTENYEKIISFFTEIYKKCAAKHSNILEVKVKALDS
  KIEKLDLIWILHDYLENRGFFYDLEEENVADKYEGIEHPSILLYDFFKKNGFFKSNSSIPKDLG
  GYSFSNLQWVNEIKKLFEVQEINPEFSEKFLNLFTSVRDYAKGPGSEHSASEYGIFQKDEKGKV
  FKKYDNIWDKTIGKCSFFVEENRSPVNYPSYEIFNLLNQLINLSTDLKTTNKKIWQLSSNDRNE
  LLDELLKVKEKAKIISISLKKNEIKKIILKDFGFEKSDIDDQDTIEGRKIIKEEPTTKLEVTKH
  LLATIYSHSSDSNWININNILEFLPYLDAICIILDREKSRGQDEVLKKLTEKNIFEVLKIDREK
  QLDFVKSIFSNTKFNFKKIGNFSLKAIREFLPKMFEQNKNSEYLKWKDEEIRRKWEEQKSKLGK
  TDKKTKYLNPRIFQDEIISPGTKNTFEQAVLVLNQIIKKYSKENIIDAIIIESPREKNDKKTIE
  EIKKRNKKGKGKTLEKLFQILNLENKGYKLSDLETKPAKLLDRLRFYHQQDGIDLYTLDKINID
  QLINGSQKYEIEHIIPYSMSYDNSQANKILTEKAENLKKGKLIASEYIKRNGDEFYNKYYEKAK
  ELFINKYKKNKKLDSYVDLDEDSAKNRFRFLTLQDYDEFQVEFLARNLNDTRYSTKLFYHALVE
  HFENNEFFTYIDENSSKHKVKISTIKGHVTKYFRAKPVQKNNGPNENLNNNKPEKIEKNRENNE
  HHAVDAAIVAIIGNKNPQIANLLTLADNKTDKKFLLHDENYKENIETGELVKIPKFEVDKLAKV
  EDLKKIIQEKYEEAKKHTAIKFSRKTRTILNGGLSDETLYGFKYDEKEDKYFKIIKKKLVTSKN
  EELKKYFENPFGKKADGKSEYTVLMAQSHLSEFNKLKEIFEKYNGFSNKTGNAFVEYMNDLALK
  EPTLKAEIESAKSVEKLLYYNFKPSDQFTYHDNINNKSFKRFYKNIRIIEYKSIPIKFKILSKH
  DGGKSFKDTLFSLYSLVYKVYENGKESYKSIPVTSQMRNFGIDEFDFLDENLYNKEKLDIYKSD
  FAKPIPVNCKPVFVLKKGSILKKKSLDIDDFKETKETEEGNYYFISTISKRFNRDTAYGLKPLK
  LSVVKPVAEPSTNPIFKEYIPIHLDELGNEYPVKIKEHTDDEKLMCTIK

• Nucleic Acids Encoding Cas9 Molecules
• Nucleic acids encoding the Cas9 molecules or Cas9 polypeptides, e.g., an eaCas9 molecule or eaCas9 polypeptides are provided herein.
• Exemplary nucleic acids encoding Cas9 molecules or Cas9 polypeptides are described in Cong et al., SCIENCE 2013, 399(6121):819-823; Wang et al., CELL 2013, 153(4):910-918; Mali et al., SCIENCE 2013, 399(6121):823-826; Jinek et al., SCIENCE 2012, 337(6096):816-821. Another exemplary nucleic acid encoding a Cas9 molecule or Cas9 polypeptide is shown in FIG. 8.
• In an embodiment, a nucleic acid encoding a Cas9 molecule or Cas9 polypeptide can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified, e.g., as described in Section VIII. In an embodiment, the Cas9 mRNA has one or more (e.g., all of the following properties: it is capped, polyadenylated, substituted with 5-methylcytidine and/or pseudouridine.
• In addition, or alternatively, the synthetic nucleic acid sequence can be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein.
• In addition, or alternatively, a nucleic acid encoding a Cas9 molecule or Cas9 polypeptide may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.
• Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S. pyogenes.

• (SEQ ID NO: 22)

  	ATGGATAAAA AGTACAGCAT CGGGCTGGAC ATCGGTACAA
  	ACTCAGTGGG GTGGGCCGTG ATTACGGACG AGTACAAGGT
  	ACCCTCCAAA AAATTTAAAG TGCTGGGTAA CACGGACAGA
  	CACTCTATAA AGAAAAATCT TATTGGAGCC TTGCTGTTCG
  	ACTCAGGCGA GACAGCCGAA GCCACAAGGT TGAAGCGGAC
  	CGCCAGGAGG CGGTATACCA GGAGAAAGAA CCGCATATGC
  	TACCTGCAAG AAATCTTCAG TAACGAGATG GCAAAGGTTG
  	ACGATAGCTT TTTCCATCGC CTGGAAGAAT CCTTTCTTGT
  	TGAGGAAGAC AAGAAGCACG AACGGCACCC CATCTTTGGC
  	AATATTGTCG ACGAAGTGGC ATATCACGAA AAGTACCCGA
  	CTATCTACCA CCTCAGGAAG AAGCTGGTGG ACTCTACCGA
  	TAAGGCGGAC CTCAGACTTA TTTATTTGGC ACTCGCCCAC
  	ATGATTAAAT TTAGAGGACA TTTCTTGATC GAGGGCGACC
  	TGAACCCGGA CAACAGTGAC GTCGATAAGC TGTTCATCCA
  	ACTTGTGCAG ACCTACAATC AACTGTTCGA AGAAAACCCT
  	ATAAATGCTT CAGGAGTCGA CGCTAAAGCA ATCCTGTCCG
  	CGCGCCTCTC AAAATCTAGA AGACTTGAGA ATCTGATTGC
  	TCAGTTGCCC GGGGAAAAGA AAAATGGATT GTTTGGCAAC
  	CTGATCGCCC TCAGTCTCGG ACTGACCCCA AATTTCAAAA
  	GTAACTTCGA CCTGGCCGAA GACGCTAAGC TCCAGCTGTC
  	CAAGGACACA TACGATGACG ACCTCGACAA TCTGCTGGCC
  	CAGATTGGGG ATCAGTACGC CGATCTCTTT TTGGCAGCAA
  	AGAACCTGTC CGACGCCATC CTGTTGAGCG ATATCTTGAG
  	AGTGAACACC GAAATTACTA AAGCACCCCT TAGCGCATCT
  	ATGATCAAGC GGTACGACGA GCATCATCAG GATCTGACCC
  	TGCTGAAGGC TCTTGTGAGG CAACAGCTCC CCGAAAAATA
  	CAAGGAAATC TTCTTTGACC AGAGCAAAAA CGGCTACGCT
  	GGCTATATAG ATGGTGGGGC CAGTCAGGAG GAATTCTATA
  	AATTCATCAA GCCCATTCTC GAGAAAATGG ACGGCACAGA
  	GGAGTTGCTG GTCAAACTTA ACAGGGAGGA CCTGCTGCGG
  	AAGCAGCGGA CCTTTGACAA CGGGTCTATC CCCCACCAGA
  	TTCATCTGGG CGAACTGCAC GCAATCCTGA GGAGGCAGGA
  	GGATTTTTAT CCTTTTCTTA AAGATAACCG CGAGAAAATA
  	GAAAAGATTC TTACATTCAG GATCCCGTAC TACGTGGGAC
  	CTCTCGCCCG GGGCAATTCA CGGTTTGCCT GGATGACAAG
  	GAAGTCAGAG GAGACTATTA CACCTTGGAA CTTCGAAGAA
  	GTGGTGGACA AGGGTGCATC TGCCCAGTCT TTCATCGAGC
  	GGATGACAAA TTTTGACAAG AACCTCCCTA ATGAGAAGGT
  	GCTGCCCAAA CATTCTCTGC TCTACGAGTA CTTTACCGTC
  	TACAATGAAC TGACTAAAGT CAAGTACGTC ACCGAGGGAA
  	TGAGGAAGCC GGCATTCCTT AGTGGAGAAC AGAAGAAGGC
  	GATTGTAGAC CTGTTGTTCA AGACCAACAG GAAGGTGACT
  	GTGAAGCAAC TTAAAGAAGA CTACTTTAAG AAGATCGAAT
  	GTTTTGACAG TGTGGAAATT TCAGGGGTTG AAGACCGCTT
  	CAATGCGTCA TTGGGGACTT ACCATGATCT TCTCAAGATC
  	ATAAAGGACA AAGACTTCCT GGACAACGAA GAAAATGAGG
  	ATATTCTCGA AGACATCGTC CTCACCCTGA CCCTGTTCGA
  	AGACAGGGAA ATGATAGAAG AGCGCTTGAA AACCTATGCC
  	CACCTCTTCG ACGATAAAGT TATGAAGCAG CTGAAGCGCA
  	GGAGATACAC AGGATGGGGA AGATTGTCAA GGAAGCTGAT
  	CAATGGAATT AGGGATAAAC AGAGTGGCAA GACCATACTG
  	GATTTCCTCA AATCTGATGG CTTCGCCAAT AGGAACTTCA
  	TGCAACTGAT TCACGATGAC TCTCTTACCT TCAAGGAGGA
  	CATTCAAAAG GCTCAGGTGA GCGGGCAGGG AGACTCCCTT
  	CATGAACACA TCGCGAATTT GGCAGGTTCC CCCGCTATTA
  	AAAAGGGCAT CCTTCAAACT GTCAAGGTGG TGGATGAATT
  	GGTCAAGGTA ATGGGCAGAC ATAAGCCAGA AAATATTGTG
  	ATCGAGATGG CCCGCGAAAA CCAGACCACA CAGAAGGGCC
  	AGAAAAATAG TAGAGAGCGG ATGAAGAGGA TCGAGGAGGG
  	CATCAAAGAG CTGGGATCTC AGATTCTCAA AGAACACCCC
  	GTAGAAAACA CACAGCTGCA GAACGAAAAA TTGTACTTGT
  	ACTATCTGCA GAACGGCAGA GACATGTACG TCGACCAAGA
  	ACTTGATATT AATAGACTGT CCGACTATGA CGTAGACCAT
  	ATCGTGCCCC AGTCCTTCCT GAAGGACGAC TCCATTGATA
  	ACAAAGTCTT GACAAGAAGC GACAAGAACA GGGGTAAAAG
  	TGATAATGTG CCTAGCGAGG AGGTGGTGAA AAAAATGAAG
  	AACTACTGGC GACAGCTGCT TAATGCAAAG CTCATTACAC
  	AACGGAAGTT CGATAATCTG ACGAAAGCAG AGAGAGGTGG
  	CTTGTCTGAG TTGGACAAGG CAGGGTTTAT TAAGCGGCAG
  	CTGGTGGAAA CTAGGCAGAT CACAAAGCAC GTGGCGCAGA
  	TTTTGGACAG CCGGATGAAC ACAAAATACG ACGAAAATGA
  	TAAACTGATA CGAGAGGTCA AAGTTATCAC GCTGAAAAGC
  	AAGCTGGTGT CCGATTTTCG GAAAGACTTC CAGTTCTACA
  	AAGTTCGCGA GATTAATAAC TACCATCATG CTCACGATGC
  	GTACCTGAAC GCTGTTGTCG GGACCGCCTT GATAAAGAAG
  	TACCCAAAGC TGGAATCCGA GTTCGTATAC GGGGATTACA
  	AAGTGTACGA TGTGAGGAAA ATGATAGCCA AGTCCGAGCA
  	GGAGATTGGA AAGGCCACAG CTAAGTACTT CTTTTATTCT
  	AACATCATGA ATTTTTTTAA GACGGAAATT ACCCTGGCCA
  	ACGGAGAGAT CAGAAAGCGG CCCCTTATAG AGACAAATGG
  	TGAAACAGGT GAAATCGTCT GGGATAAGGG CAGGGATTTC
  	GCTACTGTGA GGAAGGTGCT GAGTATGCCA CAGGTAAATA
  	TCGTGAAAAA AACCGAAGTA CAGACCGGAG GATTTTCCAA
  	GGAAAGCATT TTGCCTAAAA GAAACTCAGA CAAGCTCATC
  	GCCCGCAAGA AAGATTGGGA CCCTAAGAAA TACGGGGGAT
  	TTGACTCACC CACCGTAGCC TATTCTGTGC TGGTGGTAGC
  	TAAGGTGGAA AAAGGAAAGT CTAAGAAGCT GAAGTCCGTG
  	AAGGAACTCT TGGGAATCAC TATCATGGAA AGATCATCCT
  	TTGAAAAGAA CCCTATCGAT TTCCTGGAGG CTAAGGGTTA
  	CAAGGAGGTC AAGAAAGACC TCATCATTAA ACTGCCAAAA
  	TACTCTCTCT TCGAGCTGGA AAATGGCAGG AAGAGAATGT
  	TGGCCAGCGC CGGAGAGCTG CAAAAGGGAA ACGAGCTTGC
  	TCTGCCCTCC AAATATGTTA ATTTTCTCTA TCTCGCTTCC
  	CACTATGAAA AGCTGAAAGG GTCTCCCGAA GATAACGAGC
  	AGAAGCAGCT GTTCGTCGAA CAGCACAAGC ACTATCTGGA
  	TGAAATAATC GAACAAATAA GCGAGTTCAG CAAAAGGGTT
  	ATCCTGGCGG ATGCTAATTT GGACAAAGTA CTGTCTGCTT
  	ATAACAAGCA CCGGGATAAG CCTATTAGGG AACAAGCCGA
  	GAATATAATT CACCTCTTTA CACTCACGAA TCTCGGAGCC
  	CCCGCCGCCT TCAAATACTT TGATACGACT ATCGACCGGA
  	AACGGTATAC CAGTACCAAA GAGGTCCTCG ATGCCACCCT
  	CATCCACCAG TCAATTACTG GCCTGTACGA AACACGGATC
  	GACCTCTCTC AACTGGGCGG CGACTAG

• Provided below is the corresponding amino acid sequence of a S. pyogenes Cas9 molecule.

• (SEQ ID NO: 23)

  MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

  LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

  LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

  LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

  INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

  NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

  LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

  FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

  KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

  YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

  NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

  LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

  IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

  LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

  SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

  MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

  VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD

  SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

  TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

  REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

  YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

  TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

  QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE

  KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

  YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE

  DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

  PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ

  SITGLYETRIDLSQLGGD*

• Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of N. meningitides.

• SEQ ID NO: 24)

  ATGGCCGCCTTCAAGCCCAACCCCATCAACTACATCCTGGGCCTGGACAT

  CGGCATCGCCAGCGTGGGCTGGGCCATGGTGGAGATCGACGAGGACGAGA

  ACCCCATCTGCCTGATCGACCTGGGTGTGCGCGTGTTCGAGCGCGCTGAG

  GTGCCCAAGACTGGTGACAGTCTGGCTATGGCTCGCCGGCTTGCTCGCTC

  TGTTCGGCGCCTTACTCGCCGGCGCGCTCACCGCCTTCTGCGCGCTCGCC

  GCCTGCTGAAGCGCGAGGGTGTGCTGCAGGCTGCCGACTTCGACGAGAAC

  GGCCTGATCAAGAGCCTGCCCAACACTCCTTGGCAGCTGCGCGCTGCCGC

  TCTGGACCGCAAGCTGACTCCTCTGGAGTGGAGCGCCGTGCTGCTGCACC

  TGATCAAGCACCGCGGCTACCTGAGCCAGCGCAAGAACGAGGGCGAGACC

  GCCGACAAGGAGCTGGGTGCTCTGCTGAAGGGCGTGGCCGACAACGCCCA

  CGCCCTGCAGACTGGTGACTTCCGCACTCCTGCTGAGCTGGCCCTGAACA

  AGTTCGAGAAGGAGAGCGGCCACATCCGCAACCAGCGCGGCGACTACAGC

  CACACCTTCAGCCGCAAGGACCTGCAGGCCGAGCTGATCCTGCTGTTCGA

  GAAGCAGAAGGAGTTCGGCAACCCCCACGTGAGCGGCGGCCTGAAGGAGG

  GCATCGAGACCCTGCTGATGACCCAGCGCCCCGCCCTGAGCGGCGACGCC

  GTGCAGAAGATGCTGGGCCACTGCACCTTCGAGCCAGCCGAGCCCAAGGC

  CGCCAAGAACACCTACACCGCCGAGCGCTTCATCTGGCTGACCAAGCTGA

  ACAACCTGCGCATCCTGGAGCAGGGCAGCGAGCGCCCCCTGACCGACACC

  GAGCGCGCCACCCTGATGGACGAGCCCTACCGCAAGAGCAAGCTGACCTA

  CGCCCAGGCCCGCAAGCTGCTGGGTCTGGAGGACACCGCCTTCTTCAAGG

  GCCTGCGCTACGGCAAGGACAACGCCGAGGCCAGCACCCTGATGGAGATG

  AAGGCCTACCACGCCATCAGCCGCGCCCTGGAGAAGGAGGGCCTGAAGGA

  CAAGAAGAGTCCTCTGAACCTGAGCCCCGAGCTGCAGGACGAGATCGGCA

  CCGCCTTCAGCCTGTTCAAGACCGACGAGGACATCACCGGCCGCCTGAAG

  GACCGCATCCAGCCCGAGATCCTGGAGGCCCTGCTGAAGCACATCAGCTT

  CGACAAGTTCGTGCAGATCAGCCTGAAGGCCCTGCGCCGCATCGTGCCCC

  TGATGGAGCAGGGCAAGCGCTACGACGAGGCCTGCGCCGAGATCTACGGC

  GACCACTACGGCAAGAAGAACACCGAGGAGAAGATCTACCTGCCTCCTAT

  CCCCGCCGACGAGATCCGCAACCCCGTGGTGCTGCGCGCCCTGAGCCAGG

  CCCGCAAGGTGATCAACGGCGTGGTGCGCCGCTACGGCAGCCCCGCCCGC

  ATCCACATCGAGACCGCCCGCGAGGTGGGCAAGAGCTTCAAGGACCGCAA

  GGAGATCGAGAAGCGCCAGGAGGAGAACCGCAAGGACCGCGAGAAGGCCG

  CCGCCAAGTTCCGCGAGTACTTCCCCAACTTCGTGGGCGAGCCCAAGAGC

  AAGGACATCCTGAAGCTGCGCCTGTACGAGCAGCAGCACGGCAAGTGCCT

  GTACAGCGGCAAGGAGATCAACCTGGGCCGCCTGAACGAGAAGGGCTACG

  TGGAGATCGACCACGCCCTGCCCTTCAGCCGCACCTGGGACGACAGCTTC

  AACAACAAGGTGCTGGTGCTGGGCAGCGAGAACCAGAACAAGGGCAACCA

  GACCCCCTACGAGTACTTCAACGGCAAGGACAACAGCCGCGAGTGGCAGG

  AGTTCAAGGCCCGCGTGGAGACCAGCCGCTTCCCCCGCAGCAAGAAGCAG

  CGCATCCTGCTGCAGAAGTTCGACGAGGACGGCTTCAAGGAGCGCAACCT

  GAACGACACCCGCTACGTGAACCGCTTCCTGTGCCAGTTCGTGGCCGACC

  GCATGCGCCTGACCGGCAAGGGCAAGAAGCGCGTGTTCGCCAGCAACGGC

  CAGATCACCAACCTGCTGCGCGGCTTCTGGGGCCTGCGCAAGGTGCGCGC

  CGAGAACGACCGCCACCACGCCCTGGACGCCGTGGTGGTGGCCTGCAGCA

  CCGTGGCCATGCAGCAGAAGATCACCCGCTTCGTGCGCTACAAGGAGATG

  AACGCCTTCGACGGTAAAACCATCGACAAGGAGACCGGCGAGGTGCTGCA

  CCAGAAGACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGGTGA

  TGATCCGCGTGTTCGGCAAGCCCGACGGCAAGCCCGAGTTCGAGGAGGCC

  GACACCCCCGAGAAGCTGCGCACCCTGCTGGCCGAGAAGCTGAGCAGCCG

  CCCTGAGGCCGTGCACGAGTACGTGACTCCTCTGTTCGTGAGCCGCGCCC

  CCAACCGCAAGATGAGCGGTCAGGGTCACATGGAGACCGTGAAGAGCGCC

  AAGCGCCTGGACGAGGGCGTGAGCGTGCTGCGCGTGCCCCTGACCCAGCT

  GAAGCTGAAGGACCTGGAGAAGATGGTGAACCGCGAGCGCGAGCCCAAGC

  TGTACGAGGCCCTGAAGGCCCGCCTGGAGGCCCACAAGGACGACCCCGCC

  AAGGCCTTCGCCGAGCCCTTCTACAAGTACGACAAGGCCGGCAACCGCAC

  CCAGCAGGTGAAGGCCGTGCGCGTGGAGCAGGTGCAGAAGACCGGCGTGT

  GGGTGCGCAACCACAACGGCATCGCCGACAACGCCACCATGGTGCGCGTG

  GACGTGTTCGAGAAGGGCGACAAGTACTACCTGGTGCCCATCTACAGCTG

  GCAGGTGGCCAAGGGCATCCTGCCCGACCGCGCCGTGGTGCAGGGCAAGG

  ACGAGGAGGACTGGCAGCTGATCGACGACAGCTTCAACTTCAAGTTCAGC

  CTGCACCCCAACGACCTGGTGGAGGTGATCACCAAGAAGGCCCGCATGTT

  CGGCTACTTCGCCAGCTGCCACCGCGGCACCGGCAACATCAACATCCGCA

  TCCACGACCTGGACCACAAGATCGGCAAGAACGGCATCCTGGAGGGCATC

  GGCGTGAAGACCGCCCTGAGCTTCCAGAAGTACCAGATCGACGAGCTGGG

  CAAGGAGATCCGCCCCTGCCGCCTGAAGAAGCGCCCTCCTGTGCGCTAA

• Provided below is the corresponding amino acid sequence of a N. meningitides Cas9 molecule.

• (SEQ ID NO: 25)

  MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAE

  VPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDEN

  GLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGET

  ADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS

  HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDA

  VQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDT

  ERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEM

  KAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK

  DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYG

  DHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPAR

  IHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKS

  KDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF

  NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQ

  RILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNG

  QITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEM

  NAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA

  DTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSA

  KRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPA

  KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRV

  DVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFS

  LHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGI

  GVKTALSFQKYQIDELGKEIRPCRLKKRPPVR*

• Provided below is an amino acid sequence of a S. aureus Cas9 molecule.

• (SEQ ID NO: 26)

  MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSK

  RGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKL

  SEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYV

  AELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT

  YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA

  YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIA

  KEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQ

  IAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI

  NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV

  KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQ

  TNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNP

  FNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS

  YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTR

  YATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKH

  HAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEY

  KEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL

  IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDE

  KNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS

  RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEA

  KKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT

  YREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII

  KKG*

• Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S. aureus Cas9.

• (SEQ ID NO: 39)

  ATGAAAAGGAACTACATTCTGGGGCTGGACATCGGGATTACAAGCGTGGG

  GTATGGGATTATTGACTATGAAACAAGGGACGTGATCGACGCAGGCGTCA

  GACTGTTCAAGGAGGCCAACGTGGAAAACAATGAGGGACGGAGAAGCAAG

  AGGGGAGCCAGGCGCCTGAAACGACGGAGAAGGCACAGAATCCAGAGGGT

  GAAGAAACTGCTGTTCGATTACAACCTGCTGACCGACCATTCTGAGCTGA

  GTGGAATTAATCCTTATGAAGCCAGGGTGAAAGGCCTGAGTCAGAAGCTG

  TCAGAGGAAGAGTTTTCCGCAGCTCTGCTGCACCTGGCTAAGCGCCGAGG

  AGTGCATAACGTCAATGAGGTGGAAGAGGACACCGGCAACGAGCTGTCTA

  CAAAGGAACAGATCTCACGCAATAGCAAAGCTCTGGAAGAGAAGTATGTC

  GCAGAGCTGCAGCTGGAACGGCTGAAGAAAGATGGCGAGGTGAGAGGGTC

  AATTAATAGGTTCAAGACAAGCGACTACGTCAAAGAAGCCAAGCAGCTGC

  TGAAAGTGCAGAAGGCTTACCACCAGCTGGATCAGAGCTTCATCGATACT

  TATATCGACCTGCTGGAGACTCGGAGAACCTACTATGAGGGACCAGGAGA

  AGGGAGCCCCTTCGGATGGAAAGACATCAAGGAATGGTACGAGATGCTGA

  TGGGACATTGCACCTATTTTCCAGAAGAGCTGAGAAGCGTCAAGTACGCT

  TATAACGCAGATCTGTACAACGCCCTGAATGACCTGAACAACCTGGTCAT

  CACCAGGGATGAAAACGAGAAACTGGAATACTATGAGAAGTTCCAGATCA

  TCGAAAACGTGTTTAAGCAGAAGAAAAAGCCTACACTGAAACAGATTGCT

  AAGGAGATCCTGGTCAACGAAGAGGACATCAAGGGCTACCGGGTGACAAG

  CACTGGAAAACCAGAGTTCACCAATCTGAAAGTGTATCACGATATTAAGG

  ACATCACAGCACGGAAAGAAATCATTGAGAACGCCGAACTGCTGGATCAG

  ATTGCTAAGATCCTGACTATCTACCAGAGCTCCGAGGACATCCAGGAAGA

  GCTGACTAACCTGAACAGCGAGCTGACCCAGGAAGAGATCGAACAGATTA

  GTAATCTGAAGGGGTACACCGGAACACACAACCTGTCCCTGAAAGCTATC

  AATCTGATTCTGGATGAGCTGTGGCATACAAACGACAATCAGATTGCAAT

  CTTTAACCGGCTGAAGCTGGTCCCAAAAAAGGTGGACCTGAGTCAGCAGA

  AAGAGATCCCAACCACACTGGTGGACGATTTCATTCTGTCACCCGTGGTC

  AAGCGGAGCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAA

  GTACGGCCTGCCCAATGATATCATTATCGAGCTGGCTAGGGAGAAGAACA

  GCAAGGACGCACAGAAGATGATCAATGAGATGCAGAAACGAAACCGGCAG

  ACCAATGAACGCATTGAAGAGATTATCCGAACTACCGGGAAAGAGAACGC

  AAAGTACCTGATTGAAAAAATCAAGCTGCACGATATGCAGGAGGGAAAGT

  GTCTGTATTCTCTGGAGGCCATCCCCCTGGAGGACCTGCTGAACAATCCA

  TTCAACTACGAGGTCGATCATATTATCCCCAGAAGCGTGTCCTTCGACAA

  TTCCTTTAACAACAAGGTGCTGGTCAAGCAGGAAGAGAACTCTAAAAAGG

  GCAATAGGACTCCTTTCCAGTACCTGTCTAGTTCAGATTCCAAGATCTCT

  TACGAAACCTTTAAAAAGCACATTCTGAATCTGGCCAAAGGAAAGGGCCG

  CATCAGCAAGACCAAAAAGGAGTACCTGCTGGAAGAGCGGGACATCAACA

  GATTCTCCGTCCAGAAGGATTTTATTAACCGGAATCTGGTGGACACAAGA

  TACGCTACTCGCGGCCTGATGAATCTGCTGCGATCCTATTTCCGGGTGAA

  CAATCTGGATGTGAAAGTCAAGTCCATCAACGGCGGGTTCACATCTTTTC

  TGAGGCGCAAATGGAAGTTTAAAAAGGAGCGCAACAAAGGGTACAAGCAC

  CATGCCGAAGATGCTCTGATTATCGCAAATGCCGACTTCATCTTTAAGGA

  GTGGAAAAAGCTGGACAAAGCCAAGAAAGTGATGGAGAACCAGATGTTCG

  AAGAGAAGCAGGCCGAATCTATGCCCGAAATCGAGACAGAACAGGAGTAC

  AAGGAGATTTTCATCACTCCTCACCAGATCAAGCATATCAAGGATTTCAA

  GGACTACAAGTACTCTCACCGGGTGGATAAAAAGCCCAACAGAGAGCTGA

  TCAATGACACCCTGTATAGTACAAGAAAAGACGATAAGGGGAATACCCTG

  ATTGTGAACAATCTGAACGGACTGTACGACAAAGATAATGACAAGCTGAA

  AAAGCTGATCAACAAAAGTCCCGAGAAGCTGCTGATGTACCACCATGATC

  CTCAGACATATCAGAAACTGAAGCTGATTATGGAGCAGTACGGCGACGAG

  AAGAACCCACTGTATAAGTACTATGAAGAGACTGGGAACTACCTGACCAA

  GTATAGCAAAAAGGATAATGGCCCCGTGATCAAGAAGATCAAGTACTATG

  GGAACAAGCTGAATGCCCATCTGGACATCACAGACGATTACCCTAACAGT

  CGCAACAAGGTGGTCAAGCTGTCACTGAAGCCATACAGATTCGATGTCTA

  TCTGGACAACGGCGTGTATAAATTTGTGACTGTCAAGAATCTGGATGTCA

  TCAAAAAGGAGAACTACTATGAAGTGAATAGCAAGTGCTACGAAGAGGCT

  AAAAAGCTGAAAAAGATTAGCAACCAGGCAGAGTTCATCGCCTCCTTTTA

  CAACAACGACCTGATTAAGATCAATGGCGAACTGTATAGGGTCATCGGGG

  TGAACAATGATCTGCTGAACCGCATTGAAGTGAATATGATTGACATCACT

  TACCGAGAGTATCTGGAAAACATGAATGATAAGCGCCCCCCTCGAATTAT

  CAAAACAATTGCCTCTAAGACTCAGAGTATCAAAAAGTACTCAACCGACA

  TTCTGGGAAACCTGTATGAGGTGAAGAGCAAAAAGCACCCTCAGATTATC

  AAAAAGGGC

• If any of the above Cas9 sequences are fused with a peptide or polypeptide at the C-terminus, it is understood that the stop codon will be removed.
• Other Cas Molecules and Cas Polypeptides
• Various types of Cas molecules or Cas polypeptides can be used to practice the inventions disclosed herein. In some embodiments, Cas molecules of Type II Cas systems are used. In other embodiments, Cas molecules of other Cas systems are used. For example, Type I or Type III Cas molecules may be used. Exemplary Cas molecules (and Cas systems) are described, e.g., in Haft et al., PLoS COMPUTATIONAL BIOLOGY 2005, 1(6): e60 and Makarova et al., NATURE REVIEW MICROBIOLOGY 2011, 9:467-477, the contents of both references are incorporated herein by reference in their entirety. Exemplary Cas molecules (and Cas systems) are also shown in Table 13.

• TABLE 13
  Cas Systems

  			Structure of	Families (and
  			encoded	superfamily) of
  Gene	System type	Name from	protein (PDB	encoded
  name‡	or subtype	Haft et al.§	accessions)¶	protein#**	Representatives
  cas1	Type I	cas1	3GOD, 3LFX	COG1518	SERP2463, SPy1047
  	Type II		and 2YZS		and ygbT
  	Type III
  cas2	Type I	cas2	2IVY, 2I8E and	COG1343 and	SERP2462, SPy1048,
  	Type II		3EXC	COG3512	SPy1723 (N-terminal
  	Type III				domain) and ygbF
  cas3′	Type I‡‡	cas3	NA	COG1203	APE1232 and ygcB
  cas3″	Subtype I-A	NA	NA	COG2254	APE1231 and
  	Subtype I-B				BH0336
  cas4	Subtype I-A	cas4 and csa1	NA	COG1468	APE1239 and
  	Subtype I-B				BH0340
  	Subtype I-C
  	Subtype I-D
  	Subtype II-B
  cas5	Subtype I-A	cas5a, cas5d,	3KG4	COG1688	APE1234, BH0337,
  	Subtype I-B	cas5e, cas5h,		(RAMP)	devS and ygcI
  	Subtype I-C	cas5p, cas5t
  	Subtype I-E	and cmx5
  cas6	Subtype I-A	cas6 and cmx6	3I4H	COG1583 and	PF1131 and slr7014
  	Subtype I-B			COG5551
  	Subtype I-D			(RAMP)
  	Subtype III-
  	A Subtype
  	III-B
  cas6e	Subtype I-E	cse3	1WJ9	(RAMP)	ygcH
  cas6f	Subtype I-F	csy4	2XLJ	(RAMP)	y1727
  cas7	Subtype I-A	csa2, csd2,	NA	COG1857 and	devR and ygcJ
  	Subtype I-B	cse4, csh2,		COG3649
  	Subtype I-C	csp1 and cst2		(RAMP)
  	Subtype I-E
  cas8a1	Subtype I-	cmx1, cst1,	NA	BH0338-like	LA3191§§ and
  	A‡‡	csx8, csx13			PG2018§§
  		and CXXC-
  		CXXC
  cas8a2	Subtype I-	csa4 and csx9	NA	PH0918	AF0070, AF1873,
  	A‡‡				MJ0385, PF0637,
  					PH0918 and
  					SSO1401
  cas8b	Subtype I-	csh1 and	NA	BH0338-like	MTH1090 and
  	B‡‡	TM1802			TM1802
  cas8c	Subtype I-	csd1 and csp2	NA	BH0338-like	BH0338
  	C‡‡
  cas9	Type II‡‡	csn1 and csx12	NA	COG3513	FTN_0757 and
  					SPy1046
  cas10	Type III‡‡	cmr2, csm1	NA	COG1353	MTH326, Rv2823c§§
  		and csx11			and TM1794§§
  cas10d	Subtype I-	csc3	NA	COG1353	slr7011
  	D‡‡
  csy1	Subtype I-	csy1	NA	y1724-like	y1724
  	F‡‡
  csy2	Subtype I-F	csy2	NA	(RAMP)	y1725
  csy3	Subtype I-F	csy3	NA	(RAMP)	y1726
  cse1	Subtype I-	cse1	NA	YgcL-like	ygcL
  	E‡‡
  cse2	Subtype I-E	cse2	2ZCA	YgcK-like	ygcK
  csc1	Subtype I-D	csc1	NA	alr1563-like	alr1563
  				(RAMP)
  csc2	Subtype I-D	csc1 and csc2	NA	COG1337	slr7012
  				(RAMP)
  csa5	Subtype I-A	csa5	NA	AF1870	AF1870, MJ0380,
  					PF0643 and
  					SSO1398
  csn2	Subtype II-A	csn2	NA	SPy1049-like	SPy1049
  csm2	Subtype III-	csm2	NA	COG1421	MTH1081 and
  	A‡‡				SERP2460
  csm3	Subtype III-A	csc2 and csm3	NA	COG1337	MTH1080 and
  				(RAMP)	SERP2459
  csm4	Subtype III-A	csm4	NA	COG1567	MTH1079 and
  				(RAMP)	SERP2458
  csm5	Subtype III-A	csm5	NA	COG1332	MTH1078 and
  				(RAMP)	SERP2457
  csm6	Subtype III-A	APE2256 and	2WTE	COG1517	APE2256 and
  		csm6			SSO1445
  cmr1	Subtype III-B	cmr1	NA	COG1367	PF1130
  				(RAMP)
  cmr3	Subtype III-B	cmr3	NA	COG1769	PF1128
  				(RAMP)
  cmr4	Subtype III-B	cmr4	NA	COG1336	PF1126
  				(RAMP)
  cmr5	Subtype III-	cmr5	2ZOP and	COG3337	MTH324 and
  	B‡‡		2OEB		PF1125
  cmr6	Subtype III-B	cmr6	NA	COG1604	PF1124
  				(RAMP)
  csb1	Subtype I-U	GSU0053	NA	(RAMP)	Balac_1306 and
  					GSU0053
  csb2	Subtype I-	NA	NA	(RAMP)	Balac_1305 and
  	U§§				GSU0054
  csb3	Subtype I-U	NA	NA	(RAMP)	Balac_1303§§
  csx17	Subtype I-U	NA	NA	NA	Btus_2683
  csx14	Subtype I-U	NA	NA	NA	GSU0052
  csx10	Subtype I-U	csx10	NA	(RAMP)	Caur_2274
  csx16	Subtype III-U	VVA1548	NA	NA	VVA1548
  csaX	Subtype III-U	csaX	NA	NA	SSO1438
  csx3	Subtype III-U	csx3	NA	NA	AF1864
  csx1	Subtype III-U	csa3, csx1,	1XMX and	COG1517 and	MJ1666, NE0113,
  		csx2, DXTHG,	2I71	COG4006	PF1127 and TM1812
  		NE0113 and
  		TIGR02710
  csx15	Unknown	NA	NA	TTE2665	TTE2665
  csf1	Type U	csf1	NA	NA	AFE_1038
  csf2	Type U	csf2	NA	(RAMP)	AFE_1039
  csf3	Type U	csf3	NA	(RAMP)	AFE_1040
  csf4	Type U	csf4	NA	NA	AFE_1037

IV. Functional Analysis of Candidate Molecules
• Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9 molecule/gRNA molecule complexes, can be evaluated by art-known methods or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 molecule are described, e.g., in Jinek et al., SCIENCE 2012, 337(6096):816-821.
• Binding and Cleavage Assay: Testing the Endonuclease Activity of Cas9 Molecule
• The ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in a plasmid cleavage assay. In this assay, synthetic or in vitro-transcribed gRNA molecule is pre-annealed prior to the reaction by heating to 95° C. and slowly cooling down to room temperature. Native or restriction digest-linearized plasmid DNA (300 ng (˜8 nM)) is incubated for 60 min at 37° C. with purified Cas9 protein molecule (50-500 nM) and gRNA (50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl₂. The reactions are stopped with 5×DNA loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The resulting cleavage products indicate whether the Cas9 molecule cleaves both DNA strands, or only one of the two strands. For example, linear DNA products indicate the cleavage of both DNA strands. Nicked open circular products indicate that only one of the two strands is cleaved.
• Alternatively, the ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in an oligonucleotide DNA cleavage assay. In this assay, DNA oligonucleotides (10 pmol) are radiolabeled by incubating with 5 units T4 polynucleotide kinase and ˜3-6 pmol (˜20-40 mCi) [γ-32P]-ATP in 1×T4 polynucleotide kinase reaction buffer at 37° C. for 30 min, in a 50 μL reaction. After heat inactivation (65° C. for 20 min), reactions are purified through a column to remove unincorporated label. Duplex substrates (100 nM) are generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95° C. for 3 min, followed by slow cooling to room temperature. For cleavage assays, gRNA molecules are annealed by heating to 95° C. for 30 s, followed by slow cooling to room temperature. Cas9 (500 nM final concentration) is pre-incubated with the annealed gRNA molecules (500 nM) in cleavage assay buffer (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol) in a total volume of 9 μl. Reactions are initiated by the addition of 1 μl target DNA (10 nM) and incubated for 1 h at 37° C. Reactions are quenched by the addition of 20 μl of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95° C. for 5 min. Cleavage products are resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphor imaging. The resulting cleavage products indicate that whether the complementary strand, the non-complementary strand, or both, are cleaved.
• One or both of these assays can be used to evaluate the suitability of a candidate gRNA molecule or candidate Cas9 molecule.
• Binding Assay: Testing the Binding of Cas9 Molecule to Target DNA
• Exemplary methods for evaluating the binding of Cas9 molecule to target DNA are described, e.g., in Jinek et al., SCIENCE 2012; 337(6096):816-821.
• For example, in an electrophoretic mobility shift assay, target DNA duplexes are formed by mixing of each strand (10 nmol) in deionized water, heating to 95° C. for 3 min and slow cooling to room temperature. All DNAs are purified on 8% native gels containing 1×TBE. DNA bands are visualized by UV shadowing, excised, and eluted by soaking gel pieces in DEPC-treated H₂O. Eluted DNA is ethanol precipitated and dissolved in DEPC-treated H₂O. DNA samples are 5′ end labeled with [γ-32P]-ATP using T4 polynucleotide kinase for 30 min at 37° C. Polynucleotide kinase is heat denatured at 65° C. for 20 min, and unincorporated radiolabel is removed using a column. Binding assays are performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT and 10% glycerol in a total volume of 10 μl. Cas9 protein molecule is programmed with equimolar amounts of pre-annealed gRNA molecule and titrated from 100 pM to 1 μM. Radiolabeled DNA is added to a final concentration of 20 pM. Samples are incubated for 1 h at 37° C. and resolved at 4° C. on an 8% native polyacrylamide gel containing 1×TBE and 5 mM MgCl₂. Gels are dried and DNA visualized by phosphor imaging.
• Differential Scanning Flourimetry (DSF)
• The thermostability of Cas9-gRNA ribonucleoprotein (RNP) complexes can be measured via DSF. This technique measures the thermostability of a protein, which can increase under favorable conditions such as the addition of a binding RNA molecule, e.g., a gRNA.
• The assay is performed using two different protocols, one to test the best stoichiometric ratio of gRNA:Cas9 protein and another to determine the best solution conditions for RNP formation.
• To determine the best solution to form RNP complexes, a 2 uM solution of Cas9 in water+10× SYPRO Orange® (Life Technologies cat#S-6650) and dispensed into a 384 well plate. An equimolar amount of gRNA diluted in solutions with varied pH and salt is then added. After incubating at room temperature for 10′ and brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20° C. to 90° C. with a 1° increase in temperature every 10 seconds.
• The second assay consists of mixing various concentrations of gRNA with 2 uM Cas9 in optimal buffer from assay 1 above and incubating at RT for 10′ in a 384 well plate. An equal volume of optimal buffer+10× SYPRO Orange® (Life Technologies cat#S-6650) is added and the plate sealed with Microseal® B adhesive (MSB-1001). Following brief centrifugation to remove any bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler with the Bio-Rad CFX Manager software is used to run a gradient from 20° C. to 90° C. with a 1° increase in temperature every 10 seconds.
V. Genome Editing Approaches
• Described herein are methods for targeted knockout of the CCR5 gene, e.g., one or both alleles of the CCR5 gene, e.g., using one or more of the approaches or pathways described herein, e.g., using NHEJ. Described herein are also methods for targeted knockdown of the CCR5 gene.
• V.1 NHEJ Approaches for Gene Targeting
• As described herein, nuclease-induced non-homologous end-joining (NHEJ) can be used to target gene-specific knockouts. Nuclease-induced NHEJ can also be used to remove (e.g., delete) sequence insertions in a gene of interest.
• While not wishing to be bound by theory, it is believed that, in an embodiment, the genomic alterations associated with the methods described herein rely on nuclease-induced NHEJ and the error-prone nature of the NHEJ repair pathway. NHEJ repairs a double-strand break in the DNA by joining together the two ends; however, generally, the original sequence is restored only if two compatible ends, exactly as they were formed by the double-strand break, are perfectly ligated. The DNA ends of the double-strand break are frequently the subject of enzymatic processing, resulting in the addition or removal of nucleotides, at one or both strands, prior to rejoining of the ends. This results in the presence of insertion and/or deletion (indel) mutations in the DNA sequence at the site of the NHEJ repair. Two-thirds of these mutations typically alter the reading frame and, therefore, produce a non-functional protein. Additionally, mutations that maintain the reading frame, but which insert or delete a significant amount of sequence, can destroy functionality of the protein. This is locus dependent as mutations in critical functional domains are likely less tolerable than mutations in non-critical regions of the protein.
• The indel mutations generated by NHEJ are unpredictable in nature; however, at a given break site certain indel sequences are favored and are over represented in the population, likely due to small regions of microhomology. The lengths of deletions can vary widely; most commonly in the 1-50 bp range, but they can easily reach greater than 100-200 bp. Insertions tend to be shorter and often include short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells.
• Because NHEJ is a mutagenic process, it can also be used to delete small sequence motifs as long as the generation of a specific final sequence is not required. If a double-strand break is targeted near to a short target sequence, the deletion mutations caused by the NHEJ repair often span, and therefore remove, the unwanted nucleotides. For the deletion of larger DNA segments, introducing two double-strand breaks, one on each side of the sequence, can result in NHEJ between the ends with removal of the entire intervening sequence. Both of these approaches can be used to delete specific DNA sequences; however, the error-prone nature of NHEJ may still produce indel mutations at the site of repair.
• Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate NHEJ-mediated indels. NHEJ-mediated indels targeted to the early coding region of a gene of interest can be used to knockout (i.e., eliminate expression of) a gene of interest. For example, early coding region of a gene of interest includes sequence immediately following a transcription start site, within a first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp).
• Placement of Double Strand or Single Strand Breaks Relative to the Target Position
• In an embodiment, in which a gRNA and Cas9 nuclease generate a double strand break for the purpose of inducing NHEJ-mediated indels, a gRNA, e.g., a unimolecular (or chimeric) or modular gRNA molecule, is configured to position one double-strand break in close proximity to a nucleotide of the target position. In an embodiment, the cleavage site is between 0-30 bp away from the target position (e.g., less than 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position).
• In an embodiment, in which two gRNAs complexing with Cas9 nickases induce two single strand breaks for the purpose of inducing NHEJ-mediated indels, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position two single-strand breaks to provide for NHEJ repair a nucleotide of the target position. In an embodiment, the gRNAs are configured to position cuts at the same position, or within a few nucleotides of one another, on different strands, essentially mimicking a double strand break. In an embodiment, the closer nick is between 0-30 bp away from the target position (e.g., less than 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position), and the two nicks are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp). In an embodiment, the gRNAs are configured to place a single strand break on either side of a nucleotide of the target position.
• Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate breaks both sides of a target position. Double strand or paired single strand breaks may be generated on both sides of a target position to remove the nucleic acid sequence between the two cuts (e.g., the region between the two breaks in deleted). In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position. In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of the target position. In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position. The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).
• V.2 Single-Strand Annealing
• Single strand annealing (SSA) is another DNA repair process that repairs a double-strand break between two repeat sequences present in a target nucleic acid. Repeat sequences utilized by the SSA pathway are generally greater than 30 nucleotides in length. Resection at the break ends occurs to reveal repeat sequences on both strands of the target nucleic acid. After resection, single strand overhangs containing the repeat sequences are coated with RPA protein to prevent the repeats sequences from inappropriate annealing, e.g., to themselves. RAD52 binds to and each of the repeat sequences on the overhangs and aligns the sequences to enable the annealing of the complementary repeat sequences. After annealing, the single-strand flaps of the overhangs are cleaved. New DNA synthesis fills in any gaps, and ligation restores the DNA duplex. As a result of the processing, the DNA sequence between the two repeats is deleted. The length of the deletion can depend on many factors including the location of the two repeats utilized, and the pathway or processivity of the resection.
• In contrast to HDR pathways, SSA does not require a template nucleic acid to alter or correct a target nucleic acid sequence. Instead, the complementary repeat sequence is utilized.
• V.3 Other DNA Repair Pathways
• SSBR (Single Strand Break Repair)
• Single-stranded breaks (SSB) in the genome are repaired by the SSBR pathway, which is a distinct mechanism from the DSB repair mechanisms discussed above. The SSBR pathway has four major stages: SSB detection, DNA end processing, DNA gap filling, and DNA ligation. A more detailed explanation is given in Caldecott, Nature Reviews Genetics 9, 619-631 (August 2008), and a summary is given here.
• In the first stage, when a SSB forms, PARP1 and/or PARP2 recognize the break and recruit repair machinery. The binding and activity of PARP1 at DNA breaks is transient and it seems to accelerate SSBr by promoting the focal accumulation or stability of SSBr protein complexes at the lesion. Arguably the most important of these SSBr proteins is XRCC1, which functions as a molecular scaffold that interacts with, stabilizes, and stimulates multiple enzymatic components of the SSBr process including the protein responsible for cleaning the DNA 3′ and 5′ ends. For instance, XRCC1 interacts with several proteins (DNA polymerase beta, PNK, and three nucleases, APE1, APTX, and APLF) that promote end processing. APE1 has endonuclease activity. APLF exhibits endonuclease and 3′ to 5′ exonuclease activities. APTX has endonuclease and 3′ to 5′ exonuclease activity.
• This end processing is an important stage of SSBR since the 3′- and/or 5′-termini of most, if not all, SSBs are ‘damaged’. End processing generally involves restoring a damaged 3′-end to a hydroxylated state and and/or a damaged 5′ end to a phosphate moiety, so that the ends become ligation-competent. Enzymes that can process damaged 3′ termini include PNKP, APE1, and TDP1. Enzymes that can process damaged 5′ termini include PNKP, DNA polymerase beta, and APTX. LIG3 (DNA ligase III) can also participate in end processing. Once the ends are cleaned, gap filling can occur.
• At the DNA gap filling stage, the proteins typically present are PARP1, DNA polymerase beta, XRCC1, FEN1 (flap endonuclease 1), DNA polymerase delta/epsilon, PCNA, and LIG1. There are two ways of gap filling, the short patch repair and the long patch repair. Short patch repair involves the insertion of a single nucleotide that is missing. At some SSBs, “gap filling” might continue displacing two or more nucleotides (displacement of up to 12 bases have been reported). FEN1 is an endonuclease that removes the displaced 5′-residues. Multiple DNA polymerases, including Pol β, are involved in the repair of SSBs, with the choice of DNA polymerase influenced by the source and type of SSB.
• In the fourth stage, a DNA ligase such as LIG1 (Ligase I) or LIG3 (Ligase III) catalyzes joining of the ends. Short patch repair uses Ligase III and long patch repair uses Ligase I.
• Sometimes, SSBR is replication-coupled. This pathway can involve one or more of CtIP, MRN, ERCC1, and FEN1. Additional factors that may promote SSBR include: aPARP, PARP1, PARP2, PARG, XRCC1, DNA polymerase b, DNA polymerase d, DNA polymerase e, PCNA, LIG1, PNK, PNKP, APE1, APTX, APLF, TDP1, LIG3, FEN1, CtIP, MRN, and ERCC1.
• MMR (Mismatch Repair)
• Cells contain three excision repair pathways: MMR, BER, and NER. The excision repair pathways have a common feature in that they typically recognize a lesion on one strand of the DNA, then exo/endonucleases remove the lesion and leave a 1-30 nucleotide gap that is sub-sequentially filled in by DNA polymerase and finally sealed with ligase. A more complete picture is given in Li, Cell Research (2008) 18:85-98, and a summary is provided here.
• Mismatch repair (MMR) operates on mispaired DNA bases.
• The MSH2/6 or MSH2/3 complexes both have ATPases activity that plays an important role in mismatch recognition and the initiation of repair. MSH2/6 preferentially recognizes base-base mismatches and identifies mispairs of 1 or 2 nucleotides, while MSH2/3 preferentially recognizes larger ID mispairs.
• hMLH1 heterodimerizes with hPMS2 to form hMutL α which possesses an ATPase activity and is important for multiple steps of MMR. It possesses a PCNA/replication factor C (RFC)-dependent endonuclease activity which plays an important role in 3′ nick-directed MMR involving EXO1. (EXO1 is a participant in both HR and MMR.) It regulates termination of mismatch-provoked excision. Ligase I is the relevant ligase for this pathway. Additional factors that may promote MMR include: EXO1, MSH2, MSH3, MSH6, MLH1, PMS2, MLH3, DNA Pol d, RPA, HMGB1, RFC, and DNA ligase I.
• Base Excision Repair (BER)
• The base excision repair (BER) pathway is active throughout the cell cycle; it is responsible primarily for removing small, non-helix-distorting base lesions from the genome. In contrast, the related Nucleotide Excision Repair pathway (discussed in the next section) repairs bulky helix-distorting lesions. A more detailed explanation is given in Caldecott, Nature Reviews Genetics 9, 619-631 (August 2008), and a summary is given here.
• Upon DNA base damage, base excision repair (BER) is initiated and the process can be simplified into five major steps: (a) removal of the damaged DNA base; (b) incision of the subsequent a basic site; (c) clean-up of the DNA ends; (d) insertion of the correct nucleotide into the repair gap; and (e) ligation of the remaining nick in the DNA backbone. These last steps are similar to the SSBR.
• In the first step, a damage-specific DNA glycosylase excises the damaged base through cleavage of the N-glycosidic bond linking the base to the sugar phosphate backbone. Then AP endonuclease-1 (APE1) or bifunctional DNA glycosylases with an associated lyase activity incised the phosphodiester backbone to create a DNA single strand break (SSB). The third step of BER involves cleaning-up of the DNA ends. The fourth step in BER is conducted by Pol that adds a new complementary nucleotide into the repair gap and in the final step XRCC1/Ligase III seals the remaining nick in the DNA backbone. This completes the short-patch BER pathway in which the majority (˜80%) of damaged DNA bases are repaired. However, if the 5′-ends in step 3 are resistant to end processing activity, following one nucleotide insertion by Pol β there is then a polymerase switch to the replicative DNA polymerases, Pol δ/ε, which then add ˜2-8 more nucleotides into the DNA repair gap. This creates a 5′-flap structure, which is recognized and excised by flap endonuclease-1 (FEN-1) in association with the processivity factor proliferating cell nuclear antigen (PCNA). DNA ligase I then seals the remaining nick in the DNA backbone and completes long-patch BER. Additional factors that may promote the BER pathway include: DNA glycosylase, APE1, Polb, Pold, Pole, XRCC1, Ligase III, FEN-1, PCNA, RECQL4, WRN, MYH, PNKP, and APTX.
• Nucleotide Excision Repair (NER)
• Nucleotide excision repair (NER) is an important excision mechanism that removes bulky helix-distorting lesions from DNA. Additional details about NER are given in Marteijn et al., Nature Reviews Molecular Cell Biology 15, 465-481 (2014), and a summary is given here. NER a broad pathway encompassing two smaller pathways: global genomic NER (GG-NER) and transcription coupled repair NER (TC-NER). GG-NER and TC-NER use different factors for recognizing DNA damage. However, they utilize the same machinery for lesion incision, repair, and ligation.
• Once damage is recognized, the cell removes a short single-stranded DNA segment that contains the lesion. Endonucleases XPF/ERCC1 and XPG (encoded by ERCC5) remove the lesion by cutting the damaged strand on either side of the lesion, resulting in a single-strand gap of 22-30 nucleotides. Next, the cell performs DNA gap filling synthesis and ligation. Involved in this process are: PCNA, RFC, DNA Pol δ, DNA Pol ε or DNA Pol κ, and DNA ligase I or XRCC1/Ligase III. Replicating cells tend to use DNA pol ε and DNA ligase I, while non-replicating cells tend to use DNA Pol δ, DNA Pol κ, and the XRCC1/Ligase III complex to perform the ligation step.
• NER can involve the following factors: XPA-G, POLH, XPF, ERCC1, XPA-G, and LIG1. Transcription-coupled NER (TC-NER) can involve the following factors: CSA, CSB, XPB, XPD, XPG, ERCC1, and TTDA. Additional factors that may promote the NER repair pathway include XPA-G, POLH, XPF, ERCC1, XPA-G, LIG1, CSA, CSB, XPA, XPB, XPC, XPD, XPF, XPG, TTDA, UVSSA, USP7, CETN2, RAD23B, UV-DDB, CAK subcomplex, RPA, and PCNA.
• Interstrand Crosslink (ICL)
• A dedicated pathway called the ICL repair pathway repairs interstrand crosslinks. Interstrand crosslinks, or covalent crosslinks between bases in different DNA strand, can occur during replication or transcription. ICL repair involves the coordination of multiple repair processes, in particular, nucleolytic activity, translesion synthesis (TLS), and HDR. Nucleases are recruited to excise the ICL on either side of the crosslinked bases, while TLS and HDR are coordinated to repair the cut strands. ICL repair can involve the following factors: endonucleases, e.g., XPF and RAD51C, endonucleases such as RAD51, translesion polymerases, e.g., DNA polymerase zeta and Rev1), and the Fanconi anemia (FA) proteins, e.g., FancJ.
• Other Pathways
• Several other DNA repair pathways exist in mammals.
• Translesion synthesis (TLS) is a pathway for repairing a single stranded break left after a defective replication event and involves translesion polymerases, e.g., DNA polζ and Rev1.
• Error-free postreplication repair (PRR) is another pathway for repairing a single stranded break left after a defective replication event.
• V.4 Targeted Knockdown
• Unlike CRISPR/Cas-mediated gene knockout, which permanently eliminates expression by mutating the gene at the DNA level, CRISPR/Cas knockdown allows for temporary reduction of gene expression through the use of artificial transcription factors. Mutating key residues in both DNA cleavage domains of the Cas9 protein (e.g. the D10A and H840A mutations) results in the generation of a catalytically inactive Cas9 (eiCas9 which is also known as dead Cas9 or dCas9) molecule. A catalytically inactive Cas9 complexes with a gRNA and localizes to the DNA sequence specified by that gRNA's targeting domain, however, it does not cleave the target DNA. Fusion of the dCas9 to an effector domain, e.g., a transcription repression domain, enables recruitment of the effector to any DNA site specified by the gRNA. Although an enzymatically inactive (eiCas9) Cas9 molecule itself can block transcription when recruited to early regions in the coding sequence, more robust repression can be achieved by fusing a transcriptional repression domain (for example KRAB, SID or ERD) to the Cas9 and recruiting it to the target knockdown position, e.g., within 1000 bp of sequence 3′ of the start codon or within 500 bp of a promoter region 5′ of the start codon of a gene. It is likely that targeting DNAseI hypersensitive sites (DHSs) of the promoter may yield more efficient gene repression or activation because these regions are more likely to be accessible to the Cas9 protein and are also more likely to harbor sites for endogenous transcription factors. Especially for gene repression, it is contemplated herein that blocking the binding site of an endogenous transcription factor would aid in downregulating gene expression. In an embodiment, one or more eiCas9 molecules may be used to block binding of one or more endogenous transcription factors. In another embodiment, an eiCas9 molecule can be fused to a chromatin modifying protein. Altering chromatin status can result in decreased expression of the target gene. One or more eiCas9 molecules fused to one or more chromatin modifying proteins may be used to alter chromatin status.
• In an embodiment, a gRNA molecule can be targeted to a known transcription response elements (e.g., promoters, enhancers, etc.), a known upstream activating sequences (UAS), and/or sequences of unknown or known function that are suspected of being able to control expression of the target DNA.
• CRISPR/Cas-mediated gene knockdown can be used to reduce expression of an unwanted allele or transcript. Contemplated herein are scenarios wherein permanent destruction of the gene is not ideal. In these scenarios, site-specific repression may be used to temporarily reduce or eliminate expression. It is also contemplated herein that the off-target effects of a Cas-repressor may be less severe than those of a Cas-nuclease as a nuclease can cleave any DNA sequence and cause mutations whereas a Cas-repressor may only have an effect if it targets the promoter region of an actively transcribed gene. However, while nuclease-mediated knockout is permanent, repression may only persist as long as the Cas-repressor is present in the cells. Once the repressor is no longer present, it is likely that endogenous transcription factors and gene regulatory elements would restore expression to its natural state.
• V.5 Examples of gRNAs in Genome Editing Methods
• gRNA molecules as described herein can be used with Cas9 molecules that generate a double strand break or a single strand break to alter the sequence of a target nucleic acid, e.g., a target position or target genetic signature. gRNA molecules useful in these methods are described below.
• In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties;
• a) it can position, e.g., when targeting a Cas9 molecule that makes double strand breaks, a double strand break (i) within 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;
• b) it has a targeting domain of at least 16 nucleotides, e.g., a targeting domain of (i) 16, (ii), 17, (iii) 18, (iv) 19, (v) 20, (vi) 21, (vii) 22, (viii) 23, (ix) 24, (x) 25, or (xi) 26 nucleotides; and
• c)
• • (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
  • (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
  • (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
  • (iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom; or
  • (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(iv).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(v).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(vi).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(vii).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(viii).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(ix).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(x).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(xi).
• In an embodiment, the gRNA is configured such that it comprises properties: a and c.
• In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(ii), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(ii), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iv), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iv), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(v), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(v), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(vi), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(vi), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(vii), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(vii), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(viii), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(viii), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(ix), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(ix), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(x), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(x), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(xi), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(xi), and c(ii).
• In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties;
• a) one or both of the gRNAs can position, e.g., when targeting a Cas9 molecule that makes single strand breaks, a single strand break within (i) 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;
• b) one or both have a targeting domain of at least 16 nucleotides, e.g., a targeting domain of (i) 16, (ii), 17, (iii) 18, (iv) 19, (v) 20, (vi) 21, (vii) 22, (viii) 23, (ix) 24, (x) 25, or (xi) 26 nucleotides; and
• c)
• • (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
  • (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
  • (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
  • (iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom; or
  • (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(iv).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(v).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(vi).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(vii).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(viii).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(ix).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(x).
• In an embodiment, the gRNA is configured such that it comprises properties: a and b(xi).
• In an embodiment, the gRNA is configured such that it comprises properties: a and c.
• In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(ii), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(ii), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iv), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iv), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(v), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(v), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(vi), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(vi), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(vii), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(vii), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(viii), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(viii), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(ix), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(ix), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(x), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(x), and c(ii).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(xi), and c(i).
• In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(xi), and c(ii).
• In an embodiment, the gRNA is used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation.
• In an embodiment, the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., the H840A.
• In an embodiment, the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H863, e.g., the N863A.
• In an embodiment, a pair of gRNAs, e.g., a pair of chimeric gRNAs, comprising a first and a second gRNA, is configured such that they comprises one or more of the following properties;
• a) one or both of the gRNAs can position, e.g., when targeting a Cas9 molecule that makes single strand breaks, a single strand break within (i) 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;
• b) one or both have a targeting domain of at least 16 nucleotides, e.g., a targeting domain of (i) 16, (ii), 17, (iii) 18, (iv) 19, (v) 20, (vi) 21, (vii) 22, (viii) 23, (ix) 24, (x) 25, or (xi) 26 nucleotides;
• c) for one or both:
• • (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
  • (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
  • (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
  • (iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; or, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom; or
  • (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain;
• d) the gRNAs are configured such that, when hybridized to target nucleic acid, they are separated by 0-50, 0-100, 0-200, at least 10, at least 20, at least 30 or at least 50 nucleotides;
• e) the breaks made by the first gRNA and second gRNA are on different strands; and
• f) the PAMs are facing outwards.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(iii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(iv).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(v).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(vi).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(vii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(viii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(ix).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(x).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(xi).
• In an embodiment, one or both of the gRNAs configured such that it comprises properties: a and c.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a, b, and c.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(i), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ii), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ii), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ii), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ii), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ii), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iii), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iv), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iv), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iv), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iv), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(iv), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(v), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(v), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(v), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(v), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(v), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vi), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vi), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vi), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vi), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vi), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vii), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vii), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vii), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vii), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(vii), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(viii), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(viii), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(viii), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(viii), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(viii), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ix), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ix), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ix), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ix), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(ix), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(x), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(x), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(x), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(x), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(x), c, d, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(xi), and c(i).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(xi), and c(ii).
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(xi), c, and d.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(xi), c, and e.
• In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a(i), b(xi), c, d, and e.
• In an embodiment, the gRNAs are used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D10A mutation.
• In an embodiment, the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., the H840A.
• In an embodiment, the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at N863, e.g., the N863A.
VI. Target Cells
• Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell, e.g., to edit a target nucleic acid, in a wide variety of cells.
• In an embodiment, a cell is manipulated by altering or editing (e.g., introducing a mutation in) the CCR5 target gene, e.g., as described herein. In an embodiment, the expression of the CCR5target gene is altered or modulated, e.g., in vivo. In another embodiment, the expression of the CCR5 target gene is altered or modulated, e.g., ex vivo.
• The Cas9 and gRNA molecules described herein can be delivered to a target cell. In an embodiment, the target cell is a circulating blood cell, e.g., a T cell (e.g., a CD4+ T cell, a CD8+ T cell, a helper T cell, a regulatory T cell, a cytotoxic T cell, a memory T cell, a T cell precursor or a natural killer T cell), a B cell (e.g., a progenitor B cell, a Pre B cell, a Pro B cell, a memory B cell, a plasma B cell), a monocyte, a megakaryocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a reticulocyte, a lymphoid progenitor cell, a myeloid progenitor cell, a gut-associated lymphoid tissue (GALT) cell, a dendritic cell, a macrophage, a microglial cell, or a hematopoietic stem cell. In an embodiment, the target cell is a bone marrow cell, (e.g., a lymphoid progenitor cell, a myeloid progenitor cell, an erythroid progenitor cell, a hematopoietic stem cell, or a mesenchymal stem cell). In an embodiment, the target cell is a CD4+ T cell. In an embodiment, the target cell is a lymphoid progenitor cell (e.g. a common lymphoid progenitor (CLP) cell). In an embodiment, the target cell is a myeloid progenitor cell (e.g. a common myeloid progenitor (CMP) cell). In an embodiment, the target cell is a hematopoietic stem cell (e.g. a long term hematopoietic stem cell (LT-HSC), a short term hematopoietic stem cell (ST-HSC), a multipotent progenitor (MPP) cell, a lineage restricted progenitor (LRP) cell).
• In an embodiment, the target cell is manipulated ex vivo by editing (e.g., introducing a mutation in) the CCR5 target gene and/or modulating the expression of the CCR5 target gene, and administered to the subject. Sources of target cells for ex vivo manipulation may include, by way of example, the subject's blood, the subject's cord blood, or the subject's bone marrow. Sources of target cells for ex vivo manipulation may also include, by way of example, heterologous donor blood, cord blood, or bone marrow.
• In an embodiment, a CD4+T cell is removed from the subject, manipulated ex vivo as described above, and the CD4+T cell is returned to the subject. In an embodiment, a lymphoid progenitor cell is removed from the subject, manipulated ex vivo as described above, and the lymphoid progenitor cell is returned to the subject. In an embodiment, a myeloid progenitor cell is removed from the subject, manipulated ex vivo as described above, and the myeloid progenitor cell is returned to the subject. In an embodiment, a hematopoietic stem cell is removed from the subject, manipulated ex vivo as described above, and the hematopoietic stem cell is returned to the subject.
• A suitable cell can also include a stem cell such as, by way of example, an embryonic stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a neuronal stem cell and a mesenchymal stem cell. In an embodiment, the cell is an induced pluripotent stem cells (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from the subject, modified to correct the mutation and differentiated into a clinically relevant cell such as e.g, a CD4+ T cell, a lymphoid progenitor cell, myeloid progenitor cell, a macrophage, dendritic cell, gut associated lymphoid tissue or a hematopoietic stem cell. In an embodiment, AAV is used to transduce the target cells, e.g., the target cells described herein.
VII. Delivery, Formulations and Routes of Administration
• The components, e.g., a Cas9 molecule and gRNA molecule can be delivered or formulated in a variety of forms, see, e.g., Tables 14 and 15. In an embodiment, one Cas9 molecule and two or more (e.g., 2, 3, 4, or more) different gRNA molecules are delivered, e.g., by an AAV vector. In an embodiment, the sequence encoding the Cas9 molecule and the sequence(s) encoding the two or more (e.g., 2, 3, 4, or more) different gRNA molecules are present on the same nucleic acid molecule, e.g., an AAV vector. When a Cas9 or gRNA component is encoded as DNA for delivery, the DNA will typically but not necessarily include a control region, e.g., comprising a promoter, to effect expression. Useful promoters for Cas9 molecule sequences include CMV, EFS, EF-1a, MSCV, PGK, CAG control promoters. In an embodiment, the promoter is a constitutive promoter. In another embodiment, the promoter is a tissue specific promoter. Useful promoters for gRNAs include H1, 7SK, tRNA, and U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding a Cas9 molecule can comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In an embodiment, the sequence encoding a Cas9 molecule comprises at least two nuclear localization signals. In an embodiment a promoter for a Cas9 molecule or a gRNA molecule can be, independently, inducible, tissue specific, or cell specific.
• Table 14 provides examples of how the components can be formulated, delivered, or administered.

• TABLE 14
  Elements

  Cas9	gRNA
  Mole-	mole-
  cule(s)	cule(s)	Comments
  DNA	DNA	In this embodiment, a Cas9 molecule, typically
  		an eaCas9 molecule, and a gRNA are transcribed
  		from DNA. In this embodiment, they are
  		encoded on separate molecules.

  DNA	In this embodiment, a Cas9 molecule, typically
  	an eaCas9 molecule, and a gRNA are transcribed
  	from DNA, here from a single molecule.

  DNA	RNA	In this embodiment, a Cas9 molecule, typically
  		an eaCas9 molecule, is transcribed from
  		DNA, and a gRNA is provided as in vitro
  		transcribed or synthesized RNA
  mRNA	RNA	In this embodiment, a Cas9 molecule, typically
  		an eaCas9 molecule, is translated from in vitro
  		transcribed mRNA, and a gRNA is provided as
  		in vitro transcribed or synthesized RNA.
  mRNA	DNA	In this embodiment, a Cas9 molecule, typically
  		an eaCas9 molecule, is translated from in vitro
  		transcribed mRNA, and a gRNA is transcribed
  		from DNA.
  Protein	DNA	In this embodiment, a Cas9 molecule, typically
  		an eaCas9 molecule, is provided as a protein,
  		and a gRNA is transcribed from DNA.
  Protein	RNA	In this embodiment, an eaCas9 molecule is
  		provided as a protein, and a gRNA is provided
  		as transcribed or synthesized RNA.

• Table 15 summarizes various delivery methods for the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, as described herein.

• TABLE 15
  	Delivery
  	into Non-	Duration		Type of
  	Dividing	of	Genome	Molecule
  Delivery Vector/Mode	Cells	Expression	Integration	Delivered
  Physical (e.g.,	YES	Transient	NO	Nucleic
  electroporation, particle gun,				Acids and
  Calcium Phosphate				Proteins
  transfection, cell compression
  or squeezing)

  Viral	Retrovirus	NO	Stable	YES	RNA
  	Lentivirus	YES	Stable	YES/NO with	RNA
  				modifications
  	Adenovirus	YES	Transient	NO	DNA
  	Adeno-	YES	Stable	NO	DNA
  	Associated
  	Virus (AAV)
  	Vaccinia Virus	YES	Very	NO	DNA
  			Transient
  	Herpes Simplex	YES	Stable	NO	DNA
  	Virus
  Non-Viral	Cationic	YES	Transient	Depends on	Nucleic
  	Liposomes			what is	Acids and
  				delivered	Proteins
  	Polymeric	YES	Transient	Depends on	Nucleic
  	Nanoparticles			what is	Acids and
  				delivered	Proteins
  Biological	Attenuated	YES	Transient	NO	Nucleic
  Non-Viral	Bacteria				Acids
  Delivery	Engineered	YES	Transient	NO	Nucleic
  Vehicles	Bacteriophages				Acids
  	Mammalian	YES	Transient	NO	Nucleic
  	Virus-like				Acids
  	Particles
  	Biological	YES	Transient	NO	Nucleic
  	liposomes:				Acids
  	Erythrocyte
  	Ghosts and
  	Exosomes

  
  DNA-Based Delivery of a Cas9 Molecule and or One or More gRNA Molecule
• Nucleic acids encoding Cas9 molecules (e.g., eaCas9 molecules) and/or gRNA molecules, can be administered to subjects or delivered into cells by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding DNA can be delivered, e.g., by vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.
• DNA encoding Cas9 molecules (e.g., eaCas9 molecules) and/or gRNA molecules can be conjugated to molecules (e.g., N-acetylgalactosamine) promoting uptake by the target cells (e.g., the target cells described herein).
• In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a vector (e.g., viral vector/virus or plasmid).
• A vector can comprise a sequence that encodes a Cas9 molecule and/or a gRNA molecule. A vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused, e.g., to a Cas9 molecule sequence. For example, ae vector can comprise a nuclear localization sequence (e.g., from SV40) fused to the sequence encoding the Cas9 molecule.
• One or more regulatory/control elements, e.g., a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and splice acceptor or donor can be included in the vectors. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., a CMV promoter). In other embodiments, the promoter is recognized by RNA polymerase III (e.g., a U6 promoter). In some embodiments, the promoter is a regulated promoter (e.g., inducible promoter). In other embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue specific promoter. In some embodiments, the promoter is a viral promoter. In other embodiments, the promoter is a non-viral promoter.
• In some embodiments, the vector or delivery vehicle is a viral vector (e.g., for generation of recombinant viruses). In some embodiments, the virus is a DNA virus (e.g., dsDNA or ssDNA virus). In other embodiments, the virus is an RNA virus (e.g., an ssRNA virus). Exemplary viral vectors/viruses include, e.g., retroviruses, lentiviruses, adenovirus, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses.
• In some embodiments, the virus infects dividing cells. In other embodiments, the virus infects non-dividing cells. In some embodiments, the virus infects both dividing and non-dividing cells. In some embodiments, the virus can integrate into the host genome. In some embodiments, the virus is engineered to have reduced immunity, e.g., in human. In some embodiments, the virus is replication-competent. In other embodiments, the virus is replication-defective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and/or packaging replaced with other genes or deleted. In some embodiments, the virus causes transient expression of the Cas9 molecule and/or the gRNA molecule. In other embodiments, the virus causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanent expression, of the Cas9 molecule and/or the gRNA molecule. The packaging capacity of the viruses may vary, e.g., from at least about 4 kb to at least about 30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.
• In an embodiment, the viral vector recognizes a specific cell type or tissue. For example, the viral vector can be pseudotyped with a different/alternative viral envelope glycoprotein; engineered with a cell type-specific receptor (e.g., genetic modification(s) of one or more viral envelope glycoproteins to incorporate a targeting ligand such as a peptide ligand, a single chain antibody, or a growth factor); and/or engineered to have a molecular bridge with dual specificities with one end recognizing a viral glycoprotein and the other end recognizing a moiety of the target cell surface (e.g., a ligand-receptor, monoclonal antibody, avidin-biotin and chemical conjugation).
• Exemplary viral vectors/viruses include, e.g., retroviruses, lentiviruses, adenovirus, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses.
• In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant retrovirus. In some embodiments, the retrovirus (e.g., Moloney murine leukemia virus) comprises a reverse transcriptase, e.g., that allows integration into the host genome. In some embodiments, the retrovirus is replication-competent. In other embodiments, the retrovirus is replication-defective, e.g., having one of more coding regions for the genes necessary for additional rounds of virion replication and packaging replaced with other genes, or deleted.
• In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant lentivirus. For example, the lentivirus is replication-defective, e.g., does not comprise one or more genes required for viral replication.
• In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant adenovirus. In some embodiments, the adenovirus is engineered to have reduced immunity in human.
• In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant AAV. In some embodiments, the AAV does not incorporate its genome into that of a host cell, e.g., a target cell as describe herein. In some embodiments, the AAV can incorporate at least part of its genome into that of a host cell, e.g., a target cell as described herein. In some embodiments, the AAV is a self-complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA. AAV serotypes that may be used in the disclosed methods, include AAV1, AAV2, modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F and/or S662V), AAV3, modified AAV3 (e.g., modifications at Y705F, Y731F and/or T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/or T492V), AAV8, AAV 8.2, AAV9, AAV rh 10, and pseudotyped AAV, such as AAV2/8, AAV2/5 and AAV2/6 can also be used in the disclosed methods. In an embodiment, an AAV capsid that can be used in the methods described herein is a capsid sequence from serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh32/33, AAV.rh43, AAV.rh64R1, or AAV7m8.
• In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered in a re-engineered AAV capsid, e.g., with 50% or greater, e.g., 60% or greater, 70% or greater, 80% or greater, 90% or greater, or 95% or greater, sequence homology with a capsid sequence from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10, AAV.rh32/33, AAV.rh43, or AAV.rh64R1.
• In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a chimeric AAV capsid. Exemplary chimeric AAV capsids include, but are not limited to, AAV9i1, AAV2i8, AAV-DJ, AAV2G9, AAV2i8G9, or AAV8G9.
• In an embodiment, the AAV is a self-complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA.
• In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a hybrid virus, e.g., a hybrid of one or more of the viruses described herein. In an embodiment, the hybrid virus is hybrid of an AAV (e.g., of any AAV serotype), with a Bocavirus, B19 virus, porcine AAV, goose AAV, feline AAV, canine AAV, or MVM.
• A Packaging cell is used to form a virus particle that is capable of infecting a target cell. Such a cell includes a 293 cell, which can package adenovirus, and a ψ2 cell or a PA317 cell, which can package retrovirus. A viral vector used in gene therapy is usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into a host or target cell (if applicable), with other viral sequences being replaced by an expression cassette encoding the protein to be expressed, eg. Cas9. For example, an AAV vector used in gene therapy typically only possesses inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and gene expression in the host or target cell. The missing viral functions can be supplied in trans by the packaging cell line and/or plasmid containing E2A, E4, and VA genes from adenovirus, and plasmid encoding Rep and Cap genes from AAV, as described in “Triple Transfection Protocol.” Henceforth, the viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. In embodiment, the viral DNA is packaged in a producer cell line, which contains E1A and/or E1B genes from adenovirus. The cell line is also infected with adenovirus as a helper. The helper virus (e.g., adenovirus or HSV) or helper plasmid promotes replication of the AAV vector and expression of AAV genes from the helper plasmid with ITRs. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
• In an embodiment, the viral vector has the ability of cell type and/or tissue type recognition. For example, the viral vector can be pseudotyped with a different/alternative viral envelope glycoprotein; engineered with a cell type-specific receptor (e.g., genetic modification of the viral envelope glycoproteins to incorporate targeting ligands such as a peptide ligand, a single chain antibody, a growth factor); and/or engineered to have a molecular bridge with dual specificities with one end recognizing a viral glycoprotein and the other end recognizing a moiety of the target cell surface (e.g., ligand-receptor, monoclonal antibody, avidin-biotin and chemical conjugation).
• In an embodiment, the viral vector achieves cell type specific expression. For example, a tissue-specific promoter can be constructed to restrict expression of the transgene (Cas 9 and gRNA) in only the target cell. The specificity of the vector can also be mediated by microRNA-dependent control of transgene expression. In an embodiment, the viral vector has increased efficiency of fusion of the viral vector and a target cell membrane. For example, a fusion protein such as fusion-competent hemagglutin (HA) can be incorporated to increase viral uptake into cells. In an embodiment, the viral vector has the ability of nuclear localization. For example, a virus that requires the breakdown of the cell wall (during cell division) and therefore will not infect a non-diving cell can be altered to incorporate a nuclear localization peptide in the matrix protein of the virus thereby enabling the transduction of non-proliferating cells.
• In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a non-vector based method (e.g., using naked DNA or DNA complexes). For example, the DNA can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, transient cell compression or squeezing (e.g., as described in Lee, et al, 2012, Nano Lett 12: 6322-27), gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.
• In an embodiment, delivery via electroporation comprises mixing the cells with the Cas9- and/or gRNA-encoding DNA in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In an embodiment, delivery via electroporation is performed using a system in which cells are mixed with the Cas9- and/or gRNA-encoding DNA in a vessel connected to a device (e.g, a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.
• In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a combination of a vector and a non-vector based method. For example, a virosome comprises a liposome combined with an inactivated virus (e.g., HIV or influenza virus), which can result in more efficient gene transfer, e.g., in a respiratory epithelial cell than either a viral or a liposomal method alone.
• In an embodiment, the delivery vehicle is a non-viral vector. In an embodiment, the non-viral vector is an inorganic nanoparticle. Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe₃MnO₂) silica The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload. In an embodiment, the non-viral vector is an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.
• Exemplary lipids for gene transfer are shown below in Table 16.

• TABLE 16
  Lipids Used for Gene Transfer

  Lipid	Abbreviation	Feature
  1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine	DOPC	Helper
  1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine	DOPE	Helper
  Cholesterol		Helper
  N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-trimethylammonium chloride	DOTMA	Cationic
  1,2-Dioleoyloxy-3-trimethylammonium-propane	DOTAP	Cationic
  Dioctadecylamidoglycylspermine	DOGS	Cationic
  N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-	GAP-DLRIE	Cationic
  propanaminium bromide
  Cetyltrimethylammonium bromide	CTAB	Cationic
  6-Lauroxyhexyl ornithinate	LHON	Cationic
  1-(2,3-Dioleoyloxypropyl)-2,4,6-trimethylpyridinium	2Oc	Cationic
  2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N-dimethyl-1-	DOSPA	Cationic
  propanaminium trifluoroacetate
  1,2-Dioleyl-3-trimethylammonium-propane	DOPA	Cationic
  N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-	MDRIE	Cationic
  propanaminium bromide
  Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide	DMRI	Cationic
  3β-[N-(N′,N′-Dimethylaminoethane)-carbamoyl]cholesterol	DC-Chol	Cationic
  Bis-guanidium-tren-cholesterol	BGTC	Cationic
  1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide	DOSPER	Cationic
  Dimethyloctadecylammonium bromide	DDAB	Cationic
  Dioctadecylamidoglicylspermidin	DSL	Cationic
  rac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]-	CLIP-1	Cationic
  dimethylammonium chloride
  rac-[2(2,3-Dihexadecyloxypropyl-	CLIP-6	Cationic
  oxymethyloxy)ethyl]trimethylammonium bromide
  Ethyldimyristoylphosphatidylcholine	EDMPC	Cationic
  1,2-Distearyloxy-N,N-dimethyl-3-aminopropane	DSDMA	Cationic
  1,2-Dimyristoyl-trimethylammonium propane	DMTAP	Cationic
  O,O′-Dimyristyl-N-lysyl aspartate	DMKE	Cationic
  1,2-Distearoyl-sn-glycero-3-ethylphosphocholine	DSEPC	Cationic
  N-Palmitoyl D-erythro-sphingosyl carbamoyl-spermine	CCS	Cationic
  N-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine	diC14-amidine	Cationic
  Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl]	DOTIM	Cationic
  imidazolinium chloride
  N1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine	CDAN	Cationic
  2-(3-[Bis(3-amino-propyl)-amino]propylamino)-N-	RPR209120	Cationic
  ditetradecylcarbamoylme-ethyl-acetamide
  1,2-dilinoleyloxy-3-dimethylaminopropane	DLinDMA	Cationic
  2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane	DLin-KC2-DMA	Cationic
  dilinoleyl-methyl-4-dimethylaminobutyrate	DLin-MC3-DMA	Cationic

• Exemplary polymers for gene transfer are shown below in Table 17.

• TABLE 17
  Polymers Used for Gene Transfer

  	Polymer	Abbreviation
  	Poly(ethylene)glycol	PEG
  	Polyethylenimine	PEI
  	Dithiobis(succinimidylpropionate)	DSP
  	Dimethyl-3,3′-dithiobispropionimidate	DTBP
  	Poly(ethyleneimine)biscarbamate	PEIC
  	Poly(L-lysine)	PLL
  	Histidine modified PLL
  	Poly(N-vinylpyrrolidone)	PVP
  	Poly(propylenimine)	PPI
  	Poly(amidoamine)	PAMAM
  	Poly(amido ethylenimine)	SS-PAEI
  	Triethylenetetramine	TETA
  	Poly(β-aminoester)
  	Poly(4-hydroxy-L-proline ester)	PHP
  	Poly(allylamine)
  	Poly(α-[4-aminobutyl]-L-glycolic acid)	PAGA
  	Poly(D,L-lactic-co-glycolic acid)	PLGA
  	Poly(N-ethyl-4-vinylpyridinium bromide)
  	Poly(phosphazene)s	PPZ
  	Poly(phosphoester)s	PPE
  	Poly(phosphoramidate)s	PPA
  	Poly(N-2-hydroxypropylmethacrylamide)	pHPMA
  	Poly (2-(dimethylamino)ethyl methacrylate)	pDMAEMA
  	Poly(2-aminoethyl propylene phosphate)	PPE-EA
  	Chitosan
  	Galactosylated chitosan
  	N-Dodacylated chitosan
  	Histone
  	Collagen
  	Dextran-spermine	D-SPM

• In an embodiment, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In an embodiment, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In an embodiment, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In an embodiment, a stimuli-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.
• In an embodiment, the delivery vehicle is a biological non-viral delivery vehicle. In an embodiment, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific tissues, bacteria having modified surface proteins to alter target tissue specificity). In an embodiment, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenic, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In an embodiment, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the “empty” particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity. In an embodiment, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes—subject (i.e., patient) derived membrane-bound nanovescicle (30-100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need of for targeting ligands).
• In an embodiment, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component described herein, are delivered. In an embodiment, the nucleic acid molecule is delivered at the same time as one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered by a different means than one or more of the components of the Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the Cas9 molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced. In an embodiment, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In an embodiment, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein.
• Delivery of RNA Encoding a Cas9 Molecule
• RNA encoding Cas9 molecules (e.g., eaCas9 molecules or eiCas9 molecules) and/or gRNA molecules, can be delivered into cells, e.g., target cells described herein, by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (e.g., as described in Lee, et al., 2012, Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Cas9-encoding and/or gRNA-encoding RNA can be conjugated to molecules to promote uptake by the target cells (e.g., target cells described herein).
• In an embodiment, delivery via electroporation comprises mixing the cells with the RNA encoding Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) and/or gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In an embodiment, delivery via electroporation is performed using a system in which cells are mixed with the RNA encoding Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) and/or gRNA molecules in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.
• Delivery Cas9 Molecule Protein
• Cas9 molecules (e.g., eaCas9 molecules or eiCas9 molecules) can be delivered into cells by art-known methods or as described herein. For example, Cas9 protein molecules can be delivered, e.g., by microinjection, electroporation, transient cell compression or squeezing (e.g., as described in Lee, et al, 2012, Nano Lett 12: 6322-27), lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA or by a gRNA. Cas9 protein can be conjugated to molecules promoting uptake by the target cells (e.g., target cells described herein).
• In an embodiment, delivery via electroporation comprises mixing the cells with the Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) with or without gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical impulses of defined duration and amplitude. In an embodiment, delivery via electroporation is performed using a system in which cells are mixed with the Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) with or without gRNA molecules in a vessel connected to a device (e.g., a pump) which feeds the mixture into a cartridge, chamber or cuvette wherein one or more electrical impulses of defined duration and amplitude are applied, after which the cells are delivered to a second vessel.
• Route of Administration
• Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intrarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. Components administered systemically may be modified or formulated to target the components to cells of the blood and bone marrow.
• Local modes of administration include, by way of example, intra-bone marrow, intrathecal, and intra-cerebroventricular routes. In an embodiment, significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intra-bone marrow) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.
• In an embodiment, components described herein are delivered by intra-bone marrow injection. Injections may be made directly into the bone marrow compartment of one or more than one bone. In an embodiment, nanoparticle or viral, e.g., AAV vector, delivery is via intra-bone marrow injection.
• Administration may be provided as a periodic bolus or as continuous infusion from an internal reservoir or from an external reservoir (for example, from an intravenous bag). Components may be administered locally, for example, by continuous release from a sustained release drug delivery device.
• In addition, components may be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material which releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems may be useful, however, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). Release systems may be natural or synthetic. However, synthetic release systems are preferred because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that components having different molecular weights are released by diffusion through or degradation of the material.
• Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non-degradable polymers include, for example: polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof.
• Poly(lactide-co-glycolide) microsphere can also be used for intraocular injection. Typically the microspheres are composed of a polymer of lactic acid and glycolic acid, which are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with components described herein.
• Bi-Modal or Differential Delivery of Components
• Separate delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, and more particularly, delivery of the components by differing modes, can enhance performance, e.g., by improving tissue specificity and safety.
• In an embodiment, the Cas9 molecule and the gRNA molecule are delivered by different modes, or as sometimes referred to herein as differential modes. Different or differential modes, as used herein, refer modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g., a Cas9 molecule or gRNA molecule. For example, the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ.
• Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., adeno-associated virus or lentivirus, delivery.
• By way of example, the components, e.g., a Cas9 molecule and a gRNA molecule, can be delivered by modes that differ in terms of resulting half-life or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In an embodiment, a gRNA molecule can be delivered by such modes. The Cas9 molecule component can be delivered by a mode which results in less persistence or less exposure to the body or a particular compartment or tissue or organ.
• More generally, in an embodiment, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.
• In an embodiment, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.
• In an embodiment, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
• In an embodiment, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmcokinetic property, e.g., distribution, persistence or exposure.
• In an embodiment, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.
• In an embodiment, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.
• In an embodiment, the first component comprises gRNA, and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gRNAs are incapable of acting in isolation. The second component, a Cas9 molecule, is delivered in a transient manner, for example as mRNA or as protein, ensuring that the full Cas9 molecule/gRNA molecule complex is only present and active for a short period of time.
• Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.
• Use of differential delivery modes can enhance performance, safety and efficacy. E.g., the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by MEW molecules. A two-part delivery system can alleviate these drawbacks.
• Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in an embodiment, a first component, e.g., a gRNA molecule is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., a Cas9 molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In an embodiment, the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In an embodiment, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In embodiment, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.
• When the Cas9 molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA molecule and the Cas9 molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only be formed in the tissue that is targeted by both vectors.
Ex Vivo Delivery
• In some embodiments, components described in Table 14 are introduced into cells which are then introduced into the subject, e.g., cells are removed from a subject, manipulated ex vivo and then introduced into the subject. Methods of introducing the components can include, e.g., any of the delivery methods described in Table 15.
VIII. Modified Nucleosides, Nucleotides, and Nucleic Acids
• Modified nucleosides and modified nucleotides can be present in nucleic acids, e.g., particularly gRNA, but also other forms of RNA, e.g., mRNA, RNAi, or siRNA. As described herein, “nucleoside” is defined as a compound containing a five-carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a derivative thereof. As described herein, “nucleotide” is defined as a nucleoside further comprising a phosphate group.
• Modified nucleosides and nucleotides can include one or more of:
• (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage;
• (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar;
• (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers;
• (iv) modification or replacement of a naturally occurring nucleobase;
• (v) replacement or modification of the ribose-phosphate backbone;
• (vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety; and
• (vii) modification of the sugar.
• The modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In an embodiment, every base of a gRNA is modified, e.g., all bases have a modified phosphate group, e.g., all are phosphorothioate groups. In an embodiment, all, or substantially all, of the phosphate groups of a unimolecular or modular gRNA molecule are replaced with phosphorothioate groups.
• In an embodiment, modified nucleotides, e.g., nucleotides having modifications as described herein, can be incorporated into a nucleic acid, e.g., a “modified nucleic acid.” In some embodiments, the modified nucleic acids comprise one, two, three or more modified nucleotides. In some embodiments, at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%) of the positions in a modified nucleic acid are a modified nucleotides.
• Unmodified nucleic acids can be prone to degradation by, e.g., cellular nucleases. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the modified nucleic acids described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward nucleases.
• In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can disrupt binding of a major groove interacting partner with the nucleic acid. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo, and also disrupt binding of a major groove interacting partner with the nucleic acid.
• Definitions of Chemical Groups
• As used herein, “alkyl” is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.
• As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.
• As used herein, “alkenyl” refers to an aliphatic group containing at least one double bond.
• As used herein, “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl.
• As used herein, “arylalkyl” or “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “arylalkyl” or “aralkyl” include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.
• As used herein, “cycloalkyl” refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.
• As used herein, “heterocyclyl” refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.
• As used herein, “heteroaryl” refers to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties include, but are not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.
• Phosphate Backbone Modifications
• The Phosphate Group
• In some embodiments, the phosphate group of a modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified nucleotide, e.g., modified nucleotide present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
• Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR₃ (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR₂ (wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that is to say that a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).
• Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl).
• The phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.
• Replacement of the Phosphate Group
• The phosphate group can be replaced by non-phosphorus containing connectors. In some embodiments, the charge phosphate group can be replaced by a neutral moiety.
• Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
• Replacement of the Ribophosphate Backbone
• Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
• Sugar Modifications
• The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group. For example, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2′ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2′-alkoxide ion. The 2′-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.
• Examples of “oxy”-2′ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_nCH₂CH₂OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the “oxy”-2′ hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2′ hydroxyl can be connected, e.g., by a C_1-6 alkylene or C_1-6 heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH₂)_n-amino, (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the “oxy”-2′ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, e.g., a PEG derivative).
• “Deoxy” modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially ds RNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_nCH₂CH₂-amino (wherein amino can be, e.g., as described herein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.
• The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide “monomer” can have an alpha linkage at the 1′ position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L-nucleosides.
• Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced with α-L-threofuranosyl-(3′→2′)).
• Modifications on the Nucleobase
• The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified nucleosides and modified nucleotides that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.
• Uracil
• In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include without limitation pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U), 5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s2U), 5-aminomethyl-2-thio-uridine (nm⁵s2U), 5-methylaminomethyl-uridine (mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s2U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U), 5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τcm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm⁵s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine 5-methyl-2-thio-uridine (m⁵s2U), 1-methyl-4-thio-pseudouridine m¹s⁴ψ) 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp³U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um), 3,2′-O-dimethyl-uridine (m³Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.
• Cytosine
• In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include without limitation 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m³C), N4-acetyl-cytidine (act), 5-formyl-cytidine (f⁵C), N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k²C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm), N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm), N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine, 2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.
• Adenine
• In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include without limitation 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenosine, 7-deaza-8-aza-adenosine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A), 2-methyl-adenosine (m²A), N6-methyl-adenosine (m⁶A), 2-methylthio-N6-methyl-adenosine (ms2 m⁶A), N6-isopentenyl-adenosine (i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A), N6-(cis-hydroxyisopentanyl)adenosine (io⁶A), 2-methylthio-N6-(cis-hydroxyisopentanyl)adenosine (ms2io⁶A), N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine (t⁶A), (t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine 2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A), N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine (hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn⁶A), N6-acetyl-adenosine (ac⁶A), 7-methyl-adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, α-thio-adenosine, 2′-O-methyl-adenosine (Am), N⁶,2′-O-dimethyl-adenosine (m⁶Am), N⁶-Methyl-2′-deoxyadenosine, N6,N6,2′-O-trimethyl-adenosine (m⁶ ₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.
• Guanine
• In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include without limitation inosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀), 7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m′G), N2-methyl-guanosine (m²G), N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m²,7G), N2, N2,7-dimethyl-guanosine (m²,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′-O-methyl-guanosine (m²Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm), 1-methyl-2′-O-methyl-guanosine (m′Gm), N2,7-dimethyl-2′-O-methyl-guanosine (m²,7Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m′Im), O⁶-phenyl-2′-deoxyinosine, 2′-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O⁶-methyl-guanosine, O⁶-Methyl-2′-deoxyguanosine, 2′-F-ara-guanosine, and 2′-F-guanosine.
• Exemplary Modified gRNAs
• In some embodiments, the modified nucleic acids can be modified gRNAs. It is to be understood that any of the gRNAs described herein can be modified in accordance with this section, including any gRNA that comprises a targeting domain from Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, or 18.
• As discussed above, transiently expressed or delivered nucleic acids can be prone to degradation by, e.g., cellular nucleases. Accordingly, in one aspect the modified gRNAs described herein can contain one or more modified nucleosides or nucleotides which introduce stability toward nucleases. While not wishing to be bound by theory it is also believed that certain modified gRNAs described herein can exhibit a reduced innate immune response when introduced into a population of cells, particularly the cells of the present invention. As noted above, the term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.
• While some of the exemplary modification discussed in this section may be included at any position within the gRNA sequence, in some embodiments, a gRNA comprises a modification at or near its 5′ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of its 5′ end). In some embodiments, a gRNA comprises a modification at or near its 3′ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of its 3′ end). In some embodiments, a gRNA comprises both a modification at or near its 5′ end and a modification at or near its 3′ end.
• In an embodiment, the 5′ end of a gRNA is modified by the inclusion of a eukaryotic mRNA cap structure or cap analog (e.g., a G(5)ppp(5)G cap analog, a m7G(5)ppp(5)G cap analog, or a 3′-O-Me-m7G(5)ppp(5)G anti reverse cap analog (ARCA)). The cap or cap analog can be included during either chemical synthesis or in vitro transcription of the gRNA.
• In an embodiment, an in vitro transcribed gRNA is modified by treatment with a phosphatase (e.g., calf intestinal alkaline phosphatase) to remove the 5′ triphosphate group.
• In an embodiment, the 3′ end of a gRNA is modified by the addition of one or more (e.g., 25-200) adenine (A) residues. The polyA tract can be contained in the nucleic acid (e.g., plasmid, PCR product, viral genome) encoding the gRNA, or can be added to the gRNA during chemical synthesis, or following in vitro transcription using a polyadenosine polymerase (e.g., E. coli Poly(A)Polymerase).
• In an embodiment, in vitro transcribed gRNA contains both a 5′ cap structure or cap analog and a 3′ polyA tract. In an embodiment, an in vitro transcribed gRNA is modified by treatment with a phosphatase (e.g., calf intestinal alkaline phosphatase) to remove the 5′ triphosphate group and comprises a 3′ polyA tract.
• In some embodiments, gRNAs can be modified at a 3′ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below:
• 
• wherein “U” can be an unmodified or modified uridine.
• In another embodiment, the 3′ terminal U can be modified with a 2′3′ cyclic phosphate as shown below:
• 
• wherein “U” can be an unmodified or modified uridine.
• In some embodiments, the gRNA molecules may contain 3′ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, e.g., uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein.
• In some embodiments, sugar-modified ribonucleotides can be incorporated into the gRNA, e.g., wherein the 2′ OH-group is replaced by a group selected from H, —OR, —R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, —SH, —SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (—CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, one or more of the nucleotides of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2′-sugar modified, such as, 2′-O-methyl, 2′-O-methoxyethyl, or 2′-Fluoro modified including, e.g., 2′-F or 2′-O-methyl, adenosine (A), 2′-F or 2′-O-methyl, cytidine (C), 2′-F or 2′-O-methyl, uridine (U), 2′-F or 2′-O-methyl, thymidine (T), 2′-F or 2′-O-methyl, guanosine (G), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.
• In some embodiments, a gRNA can include “locked” nucleic acids (LNA) in which the 2′ OH-group can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH₂)_n-amino (wherein amino can be, e.g., NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).
• In some embodiments, a gRNA can include a modified nucleotide which is multicyclic (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), or threose nucleic acid (TNA, where ribose is replaced with α-L-threofuranosyl-(3′→2′)).
• Generally, gRNA molecules include the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified gRNAs can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). Although the majority of sugar analog alterations are localized to the 2′ position, other sites are amenable to modification, including the 4′ position. In an embodiment, a gRNA comprises a 4′-S, 4′-Se or a 4′-C-aminomethyl-2′-O-Me modification.
• In some embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the gRNA. In some embodiments, 0- and N-alkylated nucleotides, e.g., N6-methyl adenosine, can be incorporated into the gRNA. In some embodiments, one or more or all of the nucleotides in a gRNA molecule are deoxynucleotides.
• miRNA Binding Sites
• microRNAs (or miRNAs) are naturally occurring cellular 19-25 nucleotide long noncoding RNAs. They bind to nucleic acid molecules having an appropriate miRNA binding site, e.g., in the 3′ UTR of an mRNA, and down-regulate gene expression. While not wishing to be bound by theory it is believed that the down regulation is either by reducing nucleic acid molecule stability or by inhibiting translation. An RNA species disclosed herein, e.g., an mRNA encoding Cas9 can comprise an miRNA binding site, e.g., in its 3′UTR. The miRNA binding site can be selected to promote down regulation of expression is a selected cell type. By way of example, the incorporation of a binding site for miR-122, a microRNA abundant in liver, can inhibit the expression of the gene of interest in the liver.
EXAMPLES
• The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.
Example 1 Evaluation of Candidate Guide RNAs (gRNAs)
• The suitability of candidate gRNAs can be evaluated as described in this example. Although described for a chimeric gRNA, the approach can also be used to evaluate modular gRNAs.
• Cloning gRNAs into Vectors
• For each gRNA, a pair of overlapping oligonucleotides is designed and obtained. Oligonucleotides are annealed and ligated into a digested vector backbone containing an upstream U6 promoter and the remaining sequence of a long chimeric gRNA. Plasmid is sequence-verified and prepped to generate sufficient amounts of transfection-quality DNA. Alternate promoters maybe used to drive in vivo transcription (e.g. H1 promoter) or for in vitro transcription (e.g., a T7 promoter).
• Cloning gRNAs in Linear dsDNA Molecule (STITCHR)
• For each gRNA, a single oligonucleotide is designed and obtained. The U6 promoter and the gRNA scaffold (e.g. including everything except the targeting domain, e.g., including sequences derived from the crRNA and tracrRNA, e.g., including a first complementarity domain; a linking domain; a second complementarity domain; a proximal domain; and a tail domain) are separately PCR amplified and purified as dsDNA molecules. The gRNA-specific oligonucleotide is used in a PCR reaction to stitch together the U6 and the gRNA scaffold, linked by the targeting domain specified in the oligonucleotide. Resulting dsDNA molecule (STITCHR product) is purified for transfection. Alternate promoters may be used to drive in vivo transcription (e.g., H1 promoter) or for in vitro transcription (e.g., T7 promoter). Any gRNA scaffold may be used to create gRNAs compatible with Cas9s from any bacterial species.
• Initial gRNA Screen
• Each gRNA to be tested is transfected, along with a plasmid expressing Cas9 and a small amount of a GFP-expressing plasmid into human cells. In preliminary experiments, these cells can be immortalized human cell lines such as 293T, K562 or U2OS. Alternatively, primary human cells may be used. In this case, cells may be relevant to the eventual therapeutic cell target (e.g., a circulating blood cell, e.g., a T cell (e.g., a CD4+ T cell, a CD8+ T cell, a helper T cell, a regulatory T cell, a cytotoxic T cell, a memory T cell, a T cell precursor or a natural killer T cell)). The use of primary cells similar to the potential therapeutic target cell population may provide important information on gene targeting rates in the context of endogenous chromatin and gene expression.
• Transfection may be performed using lipid transfection (such as Lipofectamine or Fugene) or by electroporation (such as Lonza Nucleofection). Following transfection, GFP expression can be determined either by fluorescence microscopy or by flow cytometry to confirm consistent and high levels of transfection. These preliminary transfections can comprise different gRNAs and different targeting approaches (17-mers, 20-mers, nuclease, dual-nickase, etc.) to determine which gRNAs/combinations of gRNAs give the greatest activity.
• Efficiency of cleavage with each gRNA may be assessed by measuring NHEJ-induced indel formation at the target locus by a T7E1-type assay or by sequencing. Alternatively, other mismatch-sensitive enzymes, such as Cell/Surveyor nuclease, may also be used.
• For the T7E1 assay, PCR amplicons are approximately 500-700 bp with the intended cut site placed asymmetrically in the amplicon. Following amplification, purification and size-verification of PCR products, DNA is denatured and re-hybridized by heating to 95° C. and then slowly cooling. Hybridized PCR products are then digested with T7 Endonuclease I (or other mismatch-sensitive enzyme) which recognizes and cleaves non-perfectly matched DNA. If indels are present in the original template DNA, when the amplicons are denatured and re-annealed, this results in the hybridization of DNA strands harboring different indels and therefore lead to double-stranded DNA that is not perfectly matched. Digestion products may be visualized by gel electrophoresis or by capillary electrophoresis. The fraction of DNA that is cleaved (density of cleavage products divided by the density of cleaved and uncleaved) may be used to estimate a percent NHEJ using the following equation: % NHEJ=(1−(1−fraction cleaved)^1/2). The T7E1 assay is sensitive down to about 2-5% NHEJ.
• Sequencing may be used instead of, or in addition to, the T7E1 assay. For Sanger sequencing, purified PCR amplicons are cloned into a plasmid backbone, transformed, miniprepped and sequenced with a single primer. Sanger sequencing may be used for determining the exact nature of indels after determining the NHEJ rate by T7E1.
• Sequencing may also be performed using next generation sequencing techniques. When using next generation sequencing, amplicons may be 300-500 bp with the intended cut site placed asymmetrically. Following PCR, next generation sequencing adapters and barcodes (for example Illumina multiplex adapters and indexes) may be added to the ends of the amplicon, e.g., for use in high throughput sequencing (for example on an Illumina MiSeq). This method allows for detection of very low NHEJ rates.
Example 2 Assessment of Gene Targeting by NHEJ
• The gRNAs that induce the greatest levels of NHEJ in initial tests can be selected for further evaluation of gene targeting efficiency. In this case, cells are derived from disease subjects and, therefore, harbor the relevant mutation.
• Following transfection (usually 2-3 days post-transfection) genomic DNA may be isolated from a bulk population of transfected cells and PCR may be used to amplify the target region. Following PCR, gene targeting efficiency to generate the desired mutations (either knockout of a target gene or removal of a target sequence motif) may be determined by sequencing. For Sanger sequencing, PCR amplicons may be 500-700 bp long. For next generation sequencing, PCR amplicons may be 300-500 bp long. If the goal is to knockout gene function, sequencing may be used to assess what percent of alleles have undergone NHEJ-induced indels that result in a frameshift or large deletion or insertion that would be expected to destroy gene function. If the goal is to remove a specific sequence motif, sequencing may be used to assess what percent of alleles have undergone NHEJ-induced deletions that span this sequence.
Example 3 Screening of gRNAs for CCR5
• In order to identify gRNAs with the highest on target NHEJ efficiency, 24 S. pyogenes gRNAs were selected for testing (Table 18). A DNA plasmid comprised of an exemplary gRNA (including the target region and appropriate TRACR sequence) under the control of a U6 promoter was generated by restriction enzyme cloning. This DNA template was subsequently transfected into 293 cells using Lipofectamine 3000 along with a DNA plasmid encoding the appropriate Cas9 downstream of a CMV promoter. Genomic DNA was isolated from the cells 48-72 hours post transfection. To determine the rate of modification at the CCR5 gene, the target region was amplified using a locus PCR with the following primers (CCR5 exon 3 5′ primer: TATCAAGTGTCAAGTCCAATCTATGACATC (SEQ ID NO: 5752); CCR5 exon 3 3′ primer: GGAAATTCTTCCAGAATTGATACTGACTG (SEQ ID NO: 5753). After PCR amplification, a T7E1 assay was performed on the PCR product. Briefly, this assay involves melting the PCR product followed by a re-annealing step. If gene modification has occurred, there will exist double stranded products that are not perfect matches due to some frequency of insertions or deletions. These double stranded products are sensitive to cleavage by a T7 endonuclease 1 enzyme at the site of mismatch. Therefore, the efficiency of cutting by the Cas9/gRNA complex can be determined by analyzing the amount of T7E1 cleavage. The formula that is used to provide a measure of % NHEJ from the T7E1 cutting is the following: 100*(1−((1−(fraction cleaved))̂0.5)). The results of this analysis are shown in FIG. 10.

• 	TABLE 18
  	gRNA	Targeting Domain Sequence	SEQ ID NO

  	CCR5-1	GCCUCCGCUCUACUCAC	396
  	CCR5-3	GCCGCCCAGUGGGACUU	397
  	CCR5-4	GCAUAGUGAGCCCAGAA	401
  	CCR5-6	GCCUUUUGCAGUUUAUC	409
  	CCR5-10	GACAAUCGAUAGGUACC	399
  	CCR5-13	GACAAGUGUGAUCACUU	404
  	CCR5-14	GGUACCUAUCGAUUGUC	402
  	CCR5-43	GCUGCCGCCCAGUGGGACUU	388
  	CCR5-45	GGUACCUAUCGAUUGUCAGG	394
  	CCR5-47	GCAGCAUAGUGAGCCCAGAA	393
  	CCR5-49	GUGAGUAGAGCGGAGGCAGG	395
  	CCR5-52	AUGUGUCAACUCUUGAC	398
  	CCR5-53	UUGACAGGGCUCUAUUUUAU	499
  	CCR5-54	ACAGGGCUCUAUUUUAU	5749
  	CCR5-55	UCAUCCUCCUGACAAUCGAU	477
  	CCR5-56	UCCUCCUGACAAUCGAU	5750
  	CCR5-57	CCUGACAAUCGAUAGGUACC	463
  	CCR5-58	GGUGACAAGUGUGAUCACUU	4469
  	CCR5-60	CCAGGUACCUAUCGAUUGUC	391
  	CCR5-61	ACCUAUCGAUUGUCAGG	5751
  	CCR5-62	UCAGCCUUUUGCAGUUUAUC	476
  	CCR5-64	CACAUUGAUUUUUUGGC	400
  	CCR5-65	AGUAGAGCGGAGGCAGG	442
  	CCR5-66	CCUGCCUCCGCUCUACUCAC	387

Example 4 Assessment of Gene Targeting in Hematopoietic Stem Cells
• Transplantation of autologous CD34⁺ hematopoietic stem cells (HSCs) that have been genetically modified to prevent expression of the wild-type CCR5 gene product prevents entry of the HIV virus HSC progeny that are normally susceptible to HIV infection (e.g., macrophages and CD4 T-lymphocytes). Clinically, transplantation of HSCs that contain a genetic mutation in the coding sequence for the CCR5 chemokine receptor has been shown to control HIV infection long-term (Witter et. al, New England Journal of Medicine, 2009; 360(7):692-698). Genome editing with the CRISPR/Cas9 platform precisely alters endogenous gene targets by creating an indel at the targeted cut site that can lead to knock down of gene expression at the edited locus. In this Example, genome editing in human mobilized peripheral blood CD34⁺ HSCs after co-delivery of Cas9 with gRNA targeting the CCR5 locus was evaluated to induce gene editing in CD34⁺ cells.
• Human CD34⁺ HSCs cells from mobilized peripheral blood (AllCells) were thawed into StemSpan Serum-Free Expansion Medium (SFEM™, StemCell Technologies) containing 100 ng/mL each of the following cytokines: human stem cell factor (SCF), thrombopoietin (TPO), and flt-3 ligand (FL) (all from Peprotech). Cells were grown for 3 days in a humidified incubator and 5% CO ₂ 20% O₂. On day 3, media was replaced with fresh Stemspan-SFEM™ supplemented with human SCF, TPO, FL and 40 nM of the small molecule UM171 (Xcess Bio), a human HSC self-renewal agonist which has been shown to support robust expansion of human HSCs (Fares et. al, Science, 2014; 345(6203):1509-1512). The published use of UM171 involved prolonged exposure of HSCs to the small molecule for ex vivo expansion of HSCs. In the current experiment, HSCs were exposed to UM171 for 2 hours before and 24 hours after delivery of Cas9 and gRNA plasmid DNA. This UM171 treatment protocol was based on the pilot studies that indicated acute pre-treatment with UM171 before lentivirus vector mediated gene delivery improved HSC viability compared to HSCs treated with vehicle (dimethylsulfoxide, DMSO, Sigma) alone. After the 2-hour pretreatment with UM171, 1 million CD34⁺ HSCs were Nucleofected™ with the Amaxa™ 4D Nucleofector™ device (Lonza), Program EO100 using components of the P3 Primary Cell 4D-Nucleofector Kit™ (Lonza) according to the manufacturer's instructions. Briefly, one million cells were suspended in Nucleofector™ solution and the following amounts of plasmid DNA were added to the cell suspension: 1250 ng plasmid expressing CCR5 gRNA (CCR5-43) from the human U6 promoter and 3750 ng plasmid expressing wild-type S. pyogenes Cas 9 transcriptionally regulated by the CMV promoter. After Nucleofection™, cells were plated into Stemspan-SFEM™ supplemented with SCF, TPO, FL and 40 nM UM171. After overnight incubation, HSCs were plated in Stemspan-SFEM™ plus cytokines without UM171. At 96 hours after Nucleofection™, CD34⁺ cells were counted for by trypan blue exclusion and divided into 3 portions for the following analyses: a) flow cytometry analysis for assessment of viability by co-staining with 7-Aminoactinomycin-D (7-AAD) and allophycocyanin (APC)-conjugated Annexin-V antibody (ebioscience); b) flow cytometry analysis for maintenance of HSC phenotype (after co-staining with phycoerythrin (PE)-conjugated anti-human CD34 antibody and fluorescein isothicyanate (FITC)-conjugated anti-human CD90, both from BD Bioscience; c) hematopoietic colony forming cell (CFC) analysis by plating 1500 cells in semi-solid methylcellulose based Methocult medium (StemCell Technologies) that supports differentiation of erythroid and myeloid blood cell colonies from HSCs and serves as a surrogate assay to evaluate HSC multipotency and differentiation potential ex vivo; d) genomic DNA analysis for detection of editing at the CCR5 locus. Genomic DNA was extracted from HSCs 96 hours after Nucleofection™, and CCR5 locus-specific PCR reactions were performed.
• HSCs that were Nucleofected™ with Cas9 and CCR5 gRNA plasmids after pre-treatment with UM171 exhibited >93% viability (7-AAD⁻ AnnexinV⁻) and maintained co-expression of CD34 and CD90, as determined by flow cytometry analysis (FIG. 11). In addition, the UM171-treated Nucleofected™ cells were able to divide, as there was an increase in cell number with a fold-expansion similar to the level achieved win unelectroporated HSCS (Table 19). In contrast, HSCs Nucleofected™ without UM171 pre-treatment had decreased viability and cell did not expand in culture.
• Table 19 shows that UM171 preserved CD34+ HSC viability after Nucleofection™ with wild type Cas9 and CCR5-43 gRNA plasmid DNA (96 hours)

• 	TABLE 19
  		Fold expansion of
  	Condition	CD34+ cells (96 hours)

  	No Nucleofection ™	1.6
  	Nucleofection ™ + UM171 treatment	1.5
  	Nucleofection ™ + vehicle treatment	0.6

• In order to detect indels at the CCR5 locus, T7E1 assays were performed on CCR5 locus-specific PCR products that were amplified from genomic DNA samples from Nucleofected™ CD34⁺ HSCs and then percentage of indels detected at the CCR5 locus was calculated. Twenty percent indels was detected in the genomic DNA from CD34⁺ HSCs Nucleofected™ with Cas9 and CCR5 gRNA plasmids after pre-treatment with UM171.
• To evaluate maintenance of HSC potency and differentiation potential, two weeks after plating CD34⁺ HSCs in CFC assays, hematopoietic activity was quantified based on scoring the HSC progeny by enumerating the total number of hematopoietic colony forming units (CFU) and the frequencies of specific blood cell phenotypes, including: mixed myeloid/erythroid (Granulocyte-erythroid-monocyte macrophage, CFU-GEMM), myeloid (CFU-macrophage (M), granulocyte-macrophage (CFU-GM)) and erythroid (CFU-E) colonies. CD34⁺ HSCs that were Nucleofected™ after UM171 pre-treatment maintained CFC potential compared to un-Nucleofected™ HSCs (Table 20). In contrast, CD34⁺ HSCs that were Nucleofected™ without UM171 pre-treatment had reduced CFC potential (lower total CFC counts and reduced numbers of mixed-phenotype colonies (CFU-GEMM) and erythroid colonies (CFU-E)) in comparison to un-Nucleofected™ CD34⁺ HSCs.
• Table 20 shows that UM171 preserved CD34+ HSC viability after Nucleofection™ with wild-type Cas9 and CCR5-43 gRNA plasmid DNA (two weeks).

• TABLE 20
  	Number of colony forming units per 1500
  	CD34+ HSCs plated

  Condition	E	G	M	GM	GEMM	Total

  No Nucleofection ™	64	3	88	5	11	171
  Nucleofection ™ + UM171	92	40	64	32	20	228
  Nucleofection ™ + vehicle	18	22	6	1	1	28

• Delivery of co-delivery wild-type S. pyogenes Cas9 and a single CCR5 gRNA plasmid DNA supported 20% genome editing of CD34⁺ HSCs, without loss of cell viability, multipotency, self-renewal and differentiation potential. Pre-treatment and short-term (24-hour) co-culture with the HSC self-renewal agonist UM171 was critical for maintenance of HSC survival and proliferation after Nucleofection™ with Cas9/gRNA DNA. Clinically, transplantation of HSCs that contain a genetic mutation in the CCR5 gene generated by CRISPR/Cas9 related methods can be used to achieve long term control of HIV infection.
INCORPORATION BY REFERENCE
• All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
EQUIVALENTS
• Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
• Other embodiments are within the following claims.

Claims (41)

What is claimed is:

1. A CRISPR/Cas system, comprising:

a gRNA molecule comprising a targeting domain which is complementary with a target sequence of a C-C chemokine receptor type 5 (CCR5) gene; and

a Cas9 molecule.

2. The system of claim 1, wherein said system is configured to forma double strand break or a single strand break within 500 bp, 450 bp, 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 50 bp, 25 bp, or 10 bp of a CCR5 target position, thereby altering said CCR5 gene.

3. The system of claim 2, wherein said CCR5 target position is selected from the group consisting of CCR5 target knockout positions, CCR5 target knockdown positions, CCR5 target point positions, and CCR5 target hotspot mutations.

4. The system of claim 1, wherein said Cas9 molecule is selected from the group consisting of an enzymatically active Cas9 (eaCas9) molecule, an enzymatically inactive Cas9 (eiCas9) molecule, and an eiCas9 fusion protein.

5. The system of claim 4, wherein said eaCas9 molecule comprises HNH-like domain cleavage activity but has no, or no significant, N-terminal RuvC-like domain cleavage activity.

6. The system of claim 4, wherein said eaCas9 molecule is an HNH-like domain nickase.

7. The system of claim 4, wherein said eaCas9 molecule comprises a mutation at D10.

8. The system of claim 4, wherein said eaCas9 molecule comprises N-terminal RuvC-like domain cleavage activity but has no, or no significant, HNH-like domain cleavage activity.

9. The system of claim 4, wherein said eaCas9 molecule is an N-terminal RuvC-like domain nickase.

10. The system of claim 4, wherein said eaCas9 molecule comprises a mutation at H840 or N863.

11. The system of claim 4, wherein said eiCas9 fusion protein is an eiCas9-transcription repressor domain fusion.

12. The system of claim 1, wherein said Cas9 molecule is an S. aureus Cas9 molecule, an S. pyogenes Cas9 molecule, or a N. meningitidis Cas9 molecule.

13. The system of claim 2, wherein said altering said CCR5 gene comprises knocking out said CCR5 gene, or knocking down said CCR5 gene.

14. The system of claim 1, wherein said targeting domain is configured to target a coding region or a non-coding region of said CCR5 gene, wherein said non-coding region comprises a promoter region, an enhancer region, an intron, the 3′ UTR, the 5′ UTR, or a polyadenylation signal region of said CCR5 gene; and said coding region comprises an exon of said CCR5 gene.

15. The system of claim 1, wherein said targeting domain comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences disclosed in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, and 18.

16. The system of claim 1, wherein said gRNA is a modular gRNA molecule or a chimeric gRNA molecule.

17. The system of claim 1, wherein said targeting domain has a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides.

18. The system of claim 1, wherein said gRNA molecule comprises from 5′ to 3′:

a targeting domain;

a first complementarity domain;

a linking domain;

a second complementarity domain;

a proximal domain; and

a tail domain.

19. The system of claim 18, wherein said linking domain is no more than 25 nucleotides in length.

20. The system of claim 18, wherein said proximal and tail domain, taken together, are at least 20, at least 25, at least 30, or at least 40 nucleotides in length.

21. A cell transfected with the CRISPR/Cas system of claim 1.

22. A gRNA molecule comprising a targeting domain which is complementary with a target sequence of a CCR5 gene.

23. The gRNA molecule of claim 22, wherein said targeting domain comprises or consists of a nucleotide sequence that is the same as, or differs by no more than 3 nucleotides from, a targeting domain sequence selected from the targeting domain sequences disclosed in Tables 1A-1F, 2A-2C, 3A-3E, 4A-4C, 5A-5C, 6A-6E, 7A-7C, and 18.

24. A composition comprising the gRNA molecule of claim 22.

25. The composition of claim 24, further comprising a Cas9 molecule.

26. A nucleic acid composition that comprises: (a) a first nucleotide sequence that encodes a gRNA molecule comprising a targeting domain that is complementary with a target sequence of a CCR5 gene.

27. The nucleic acid composition of claim 26, further comprising: (b) a second nucleotide sequence that encodes a Cas9 molecule.

28. The nucleic acid of claim 27, wherein said Cas9 molecule is selected from the group consisting of an eaCas9 molecule, an eiCas9 molecule, and an eiCas9 fusion protein.

29. The nucleic acid of claim 27, wherein said Cas9 molecule is an S. aureus Cas9 molecule, an S. pyogenes Cas9 molecule, or a N. meningitidis Cas9 molecule.

30. The nucleic acid composition of claim 27, wherein (a) and (b) are present on one nucleic acid molecule; or (a) is present on a first nucleic acid molecule and (b) is present on a second nucleic acid molecule.

31. The nucleic acid composition of claim 30, wherein each of said nucleic acid molecule, said first nucleic acid molecule, and said second nucleic acid molecule is a DNA plasmid.

32. The nucleic acid composition of claim 26, further comprising: (c) a third nucleotide sequence that encodes a second gRNA molecule comprising a targeting domain that is complementary with a second target sequence of said CCR5 gene.

33. A cell transfected with the nucleic acid composition of claim 26.

34. A method of altering a CCR5 gene in a cell, comprising administering to said cell:

(i) a CRISPR/Cas system comprising: (a) a gRNA molecule comprising a targeting domain which is complementary with a target domain sequence of said CCR5 gene and (b) a Cas9 molecule; or

(ii) a nucleic acid composition that comprises: (a) a first nucleotide sequence encoding a gRNA molecule comprising a targeting domain that is complementary with a target sequence of a CCR5 gene and (b) a second nucleotide sequence encoding a Cas9 molecule.

35. The method of claim 34, wherein said alteration comprises knockout of said CCR5 gene or knockdown of said CCR5 gene.

36. The method of claim 35, wherein said knockout of said CCR5 gene comprises:

(a) insertion or deletion of one or more nucleotides in close proximity to or within the early coding region of said CCR5 gene, or

(b) deletion of a genomic sequence comprising at least a portion of said CCR5 gene.

37. The method of claim 35, wherein said alteration comprises knockdown of said CCR5 gene and said Cas9 molecule is an eiCas9 molecule or an eiCas9 fusion protein.

38. The method of claim 34, wherein said alteration of said CCR5 gene results in reduction or elimination of (a) expression of said CCR5 gene, (b) CCR5 protein function, and/or (c) level of CCR5 protein.

39. The method of claim 34, wherein said cell is from a subject suffering from or at risk for HIV infection or AIDS.

40. The method of claim 34, wherein said cell is selected from the group consisting of a stem cell, a progenitor cell, a T cell, a B cell, and a blood cell.

41. The method of claim 34, wherein said cell is a hematopoietic stem cell.

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